GMG-3 GMG-4 and GMG-6 polynucleotides and polypeptides and uses thereof

ABSTRACT

The present invention relates to the field of metabolic research. Metabolic disorders, such as obesity, are a public health problem that is serious and widespread. GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides have been identified that are beneficial in the treatment of metabolic disorders. These compounds should be effective for reducing body mass and for treating metabolic-related diseases and disorders. These metabolic-related diseases and disorders include hyperlipidemias, atherosclerosis, diabetes, and hypertension.

FIELD OF THE INVENTION

The present invention relates to the field of metabolic research, inparticular the discovery of compounds effective for reducing body massand useful for treating metabolic-related diseases and disorders. Themetabolic-related diseases or disorders envisioned to be treated by themethods of the invention include, but are not limited to,hyperlipidemia, atherosclerosis, diabetes, and hypertension.

BACKGROUND OF THE INVENTION

The following discussion is intended to facilitate the understanding ofthe invention, but is not intended nor admitted to be prior art to theinvention.

Obesity is a public health problem that is serious, widespread, andincreasing. In the United States, 20 percent of the population is obese;in Europe, a slightly lower percentage is obese (Friedman (2000) Nature404:632-634). Obesity is associated with increased risk of hypertension,cardiovascular disease, diabetes, and cancer as well as respiratorycomplications and osteoarthritis (Kopelman (2000) Nature 404:635-643).Even modest weight loss ameliorates these associated conditions.

While still acknowledging that lifestyle factors including environment,diet, age and exercise play a role in obesity, twin studies, analyses offamilial aggregation, and adoption studies all indicate that obesity islargely the result of genetic factors (Barsh et al (2000) Nature404:644-651). In agreement with these studies, is the fact that anincreasing number of metabolic-related genes are being identified. Someof the more extensively studied genes include those encoding leptin (ob)and its receptor (db), pro-opiomelanocortin (Pomc),melanocortin-4-receptor (Mc4r), agouti protein (A^(y)), carboxypeptidaseE (fat), 5-hydroxytryptamine receptor 2C (Htr2c), nescient basichelix-loop-helix 2 (Nhlh2), prohormone convertase 1 (PCSK1), and tubbyprotein (tubby) (rev'd in Barsh et al (2000) Nature 404:644-651).

SUMMARY OF THE INVENTION

The instant invention is based on Genset Metabolic Genes-3, 4, and 6(GMG-3), (GMG-4), and (GMG-6). GMG-3 and GMG-4 are of human origin.Cluster 1 full-length polypeptide can be considered to be a C-terminalfragment of GMG-4 full-length polypeptide. GMG-6 is the mouse orthologueof GMG-3 and GMG-4. GMG-6A and GMG-6B correspond to splice variants ofGMG-6. GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B full-lengthpolypeptides are similar at the amino acid level to APM1, a humanprotein that has been implicated in obesity and diabetes and whichstructurally resembles TNFα. GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6Bfull-length polypeptides are comprised of a C-terminal globular C1 qhomology domain preceded by a collagen-like region. By analogy to TNFα,globular head polypeptide fragments of GMG-3, GMG-4, Cluster 1, GMG-6A,and GMG-6B comprising TVFSRNVQVSLV (SEQ ID NO:14) or the extended loopQVTGGERFNGLFAD (SEQ ID NO:15) contact receptor and to have agonistactivity. Results from Northern blot analysis and RT-PCR indicatesexpression of GMG-3 and/or GMG-4 in liver, heart, and skeletal muscle,but not in adipose tissue or brain.

The invention includes polypeptides encoded by GMG-3, GMG-4, Cluster 1,GMG-6A, and GMG-6B, which include both the full-length polypeptide andfragments thereof, preferably said polypeptide fragments comprising allor part of the C-terminal globular C1q homology domain. The GMG-3,GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptide fragments containingall or part of the C-terminal globular C1q homology domain have in vitroand in vivo biological activity as described herein, including utilityfor weight reduction, prevention of weight gain and control of bloodglucose levels in humans and other mammals. More specifically, thebiological activities of the GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6Bpolypeptides, including fragments, include reduction of elevated freefatty acid levels caused by administration of epinephrine, i.v.injection of “intralipid”, or administration of a high fat test meal, aswell as increased fatty acid oxidation in muscle cells, reduction inglucose levels, modulation of energy expenditure, resistance to insulinand weight reduction in mammals consuming a high fat/high sucrose diet.

Thus, the invention is drawn to GMG-3, GMG-4, Cluster 1, GMG-6A, andGMG-6B polypeptides, polynucleotides encoding said GMG-3, GMG-4, Cluster1, GMG-6A, and GMG-6B polypeptides, methods of using GMG-6 genomicsequence, vectors comprising said GMG-3, GMG-4, Cluster 1, GMG-6A, andGMG-6B polynucleotides, and cells recombinant for said GMG-3, GMG-4,Cluster 1, GMG-6A, and GMG-6B polynucleotides, as well as topharmaceutical and physiologically acceptable compositions comprisingsaid GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides andmethods of administering said GMG-3, GMG-4, Cluster 1, GMG-6A, andGMG-6B pharmaceutical and physiologically acceptable compositions inorder to reduce body weight or to treat metabolic-related diseases anddisorders. Assays for identifying agonists and antagonists ofmetabolic-related activity are also part of the invention.

In a first aspect, the invention features purified, isolated, orrecombinant GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptidesthat have lipid partitioning, lipid metabolism, and insulin-likeactivities. Preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6Bpolypeptide fragments are said polypeptide fragments having activity,wherein said activity is also selected from the group consisting oflipid partitioning, lipid metabolism, and insulin-like activity. Inpreferred embodiments, said polypeptide fragment comprises, consistsessentially of, or consists of, at least 6 consecutive amino acids andnot more than 333 consecutive amino acids of SEQ ID NO: 2 or 4,preferably wherein said polypeptide fragment is comprised of one or moreof amino acids 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237,238, 239, 240, 241, 242, 243, 244, 245, 245, 247, 248, 249, 250, 251,252, or 253, and more preferably wherein said polypeptide fragment iscomprised of the sequence TVFSRNVQVSLV (amino acids 256-267 of SEQ IDNO: 2 or 4) or QVTGGERFNGLFAD (amino acids 304-317 of SEQ ID NO: 2 or4); or at least 6 and not more than 225 consecutive amino acids of SEQID NO: 6, preferably wherein said polypeptide fragment is comprised ofone or more of amino acids 119, 120, 121, 122, 123, 124, 125, 126, 127,128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144, or 145, and more preferably wherein said polypeptidefragment is comprised of the sequence TVFSRNVQVSLV (amino acids 148-159of SEQ ID NO: 6) or QVTGGERFNGLFAD (amino acids 196-209 of SEQ ID NO:6); at least 6 consecutive amino acids and not more than 330 consecutiveamino acids of SEQ ID NO: 8, preferably wherein said polypeptidefragment is comprised of one or more of amino acids 224, 225, 226, 227,228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241,242, 243, 244, 245, 246, 247, 248, 249, or 250, and more preferablywherein said polypeptide fragment is comprised of the sequenceTVFSRNVQVSLV (amino acids 253-264 of SEQ ID NO: 8) or QVTGGERFNGLFAD(amino acids 301-314 of SEQ ID NO: 8); or at least 6 and not more than323 consecutive amino acids of SEQ ID NO: 10, preferably wherein saidpolypeptide fragment is comprised of one or more of amino acids 217,218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231,232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, or 243, and morepreferably wherein said polypeptide fragment is comprised of thesequence TVFSRNVQVSLV (amino acids 246-257 of SEQ ID NO: 10) orQVTGGERFNGLFAD (amino acids 294-307 of SEQ ID NO: 10).

In other preferred embodiments, GMG-3 or GMG-4 polypeptide fragmentshaving activity are selected from amino acids 20-333, 43-333, 44-333,45-333, 46-333, 47-333, 48-333, 49-333, 50-333, 51-333, 52-333, 53-333,54-333, 55-333, 56-333, 57-333, 58-333, 59-333, 60-333, 61-333, 62-333,63-333, 64-333, 65-333, 66-333, 67-333, 68-333, 69-333, 70-333, 71-333,72-333, 73-333, 74-333, 75-333, 76-333, 77-333, 78-333, 79-333, 80-333,81-333, 82-333, 83-333, 84-333, 85-333, 86-333, 87-333, 88-333, 89-333,90-333, 91-333, 92-333, 93-333, 94-333, 95-333, 96-333, 97-333, 98-333,99-333, 100-333, 101-333, 102-333, 103-333, 104-333, 105-333, 106-333,107-333, 108-333, 109-333, 110-333, 111-333, 112-333, 113-333, 114-333,115-333, 116-333, 117-333, 118-333, 119-333, 120-333, 121-333, 122-333,123-333, 124-333, 125-333, 126-333, 127-333, 128-333, 129-333, 130-333,131-333, 132-333, 133-333, 134-333, 135-333, 136-333, 137-333, 138-333,139-333, 140-333, 141-333, 142-333, 143-333, 144-333, 145-333, 146-333,147-333, 148-333, 149-333, 150-333, 151-333, 152-333, 153-333, 154-333,155-333, 156-333, 157-333, 158-333, 159-333, 160-333, 161-333, 162-333,163-333, 164-333, 165-333, 166-333, 167-333, 168-333, 169-333, 170-333,171-333, 172-333, 173-333, 174-333, 175-333, 176-333, 177-333, 178-333,179-333, 180-333, 181-333, 182-333, 183-333, 184-333, 185-333, 186-333,187-333, 188-333, 189-333, 190-333, 191-333, 192-333, 193-333, 194-333,195-333, 196-333, 197-333, 198-333, 199-333, 200-333, 201-333 or 202-333of SEQ ID NO: 2 or 4. In other preferred embodiments, Cluster 1polypeptide fragments having activity are selected from amino acids1-225, 2-225, 3-225, 4-225, 5-225, 6-225, 7-225, 8-225, 9-225, 10-225,11-225, 12-225, 13-225, 14-225, 15-225, 16-225, 17-225, 18-225, 19-225,20-225, 21-225, 22-225, 23-225, 24-225, 25-225, 26-225, 27-225, 28-225,29-225, 30-225, 31-225, 32-225, 33-225, 34-225, 35-225, 36-225, 37-225,38-225, 39-225, 40-225, 41-225, 42-225, 43-225, 44-225, 45-225, 46-225,47-225, 48-225, 49-225, 50-225, 51-225, 52-225, 53-225, 54-225, 55-225,56-225, 57-225, 58-225, 59-225, 60-225, 61-225, 62-225, 63-225, 64-225,65-225, 66-225, 67-225, 68-225, 69-225, 70-225, 71-225, 72-225, 73-225,74-225, 75-225, 76-225, 77-225, 78-225, 79-225, 80-225, 81-225, 82-225,83-225, 84-225, 85-225, 86-225, 87-225, 88-225, 89-225, 90-225, 91-225,92-225, 93-225 or 94-225 of SEQ ID NO: 6. In other preferredembodiments, GMG-6A polypeptide fragments having activity are selectedfrom 20-330, 43-330, 44-330, 45-330, 46-330, 47-330, 48-330, 49-330,50-330, 51-330, 52-330, 53-330, 54-330, 55-330, 56-330, 57-330, 58-330,59-330, 60-330, 61-330, 62-330, 63-330, 64-330, 65-330, 66-330, 67-330,68-330, 69-330, 70-330, 71-330, 72-330, 73-330, 74-330, 75-330, 76-330,77-330, 78-330, 79-330, 80-330, 81-330, 82-330, 83-330, 84-330, 85-330,86-330, 87-330, 88-330, 89-330, 90-330, 91-330, 92-330, 93-330, 94-330,95-330, 96-330, 97-330, 98-330, 99-330, 100-330, 101-330, 102-330,103-330, 104-330, 105-330, 106-330, 107-330, 108-330, 109-330, 110-330,111-330, 112-330, 113-330, 114-330, 115-330, 116-330, 117-330, 118-330,119-330, 120-330, 121-330, 122-330, 123-330, 124-330, 125-330, 126-330,127-330, 128-330, 129-330, 130-330, 131-330, 132-330, 133-330, 134-330,135-330, 136-330, 137-330, 138-330, 139-330, 140-330, 141-330, 142-330,143-330, 144-330, 145-330, 146-330, 147-330, 148-330, 149-330, 150-330,151-330, 152-330, 153-330, 154-330, 155-330, 156-330, 157-330, 158-330,159-330, 160-330, 161-330, 162-330, 163-330, 164-330, 165-330, 166-330,167-330, 168-330, 169-330, 170-330, 171-330, 172-330, 173-330, 174-330,175-330, 176-330, 177-330, 178-330, 179-330, 180-330, 181-330, 182-330,183-330, 184-330, 185-330, 186-330, 187-330, 188-330, 189-330, 190-330,191-330, 192-330, 193-330, 194-330, 195-330, 196-330, 197-330, 198-330or 199-330 of SEQ ID NO; 8. In other preferred embodiments, GMG-6Bpolypeptide fragments having activity are selected from 20-323, 43-323,44-323, 45-323, 46-323, 47-323, 48-323, 49-323, 50-323, 51-323, 52-323,53-323, 54-323, 55-323, 56-323, 57-323, 58-323, 59-323, 60-323, 61-323,62-323, 63-323, 64-323, 65-323, 66-323, 67-323, 68-323, 69-323, 70-323,71-323, 72-323, 73-323, 74-323, 75-323, 76-323, 77-323, 78-323, 79-323,80-323, 81-323, 82-323, 83-323, 84-323, 85-323, 86-323, 87-323, 88-323,89-323, 90-323, 91-323, 92-323, 93-323, 94-323, 95-323, 96-323, 97-323,98-323, 99-323, 100-323, 101-323, 102-323, 103-323, 104-323, 105-323,106-323, 107-323, 108-323, 109-323, 110-323, 111-323, 112-323, 113-323,114-323, 115-323, 116-323, 117-323, 118-323, 119-323, 120-323, 121-323,122-323, 123-323, 124-323, 125-323, 126-323, 127-323, 128-323, 129-323,130-323, 131-323, 132-323, 133-323, 134-323, 135-323, 136-323, 137-323,138-323, 139-323, 140-323, 141-323, 142-323, 143-323, 144-323, 145-323,146-323, 147-323, 148-323, 149-323, 150-323, 151-323, 152-323, 153-323,154-323, 155-323, 156-323, 157-323, 158-323, 159-323, 160-323, 161-323,162-323, 163-323, 164-323, 165-323, 166-323, 167-323, 168-323, 169-323,170-323, 171-323, 172-323, 173-323, 174-323, 175-323, 176-323, 177-323,178-323, 179-323, 180-323, 181-323, 182-323, 183-323, 184-323, 185-323,186-323, 187-323, 188-323, 189-323, 190-323, 191-323 or 192-323 of SEQID NO: 10.

In more preferred embodiments, GMG-3 or GMG-4 polypeptide fragmentshaving activity are selected from amino acids 20-333, 43-333, 45-333,46-333, 50-333, 53-333, 61-333, 67-333, 74-333, 75-333, 77-333, 81-333,82-333, 86-333, 89-333, 95-333, 100-333, 104-333, 109-333, 113-333,116-333, 125-333, 128-333, 140-333, 160-333, 164-333, 179-333, 182-333,185-333, 188-333, 191-333, 193-333, 201-333, 202-333, 227-333, 252-333,252-267, 252-317, 256-267, 256-317, or 304-317 of SEQ ID NO: 2 or 4. Inother more preferred embodiments, Cluster 1 polypeptide fragments havingactivity are selected from amino acids 1-225, 5-225, 8-225, 17-225,20-225, 32-225, 52-225, 56-225, 71-225, 74-225, 77-225, 80-225, 83-225,85-225, 93-225, 94-225, 119-225, 144-225, 144-159, 144-209, 148-159,148-209, or 196-209 of SEQ ID NO: 6. In other more preferredembodiments, GMG-6A polypeptide fragments having activity are selectedfrom amino acids 20-330, 43-330, 45-330, 46-330, 50-333, 53-330, 64-330,68-330, 71-330, 72-330, 75-330, 78-330, 79-330, 83-330, 86-330, 92-330,97-330, 101-330, 122-330, 125-330, 146-330, 157-330, 161-330, 176-330,179-330, 182-330, 185-330, 188-330, 190-330, 198-330, 199-330, 224-330,249-330, 249-264,249-314, 253-264, 253-314 or 301-314 of SEQ ID NO: 8.In other more preferred embodiments, GMG-6B polypeptide fragments havingactivity are selected from amino acids 20-323, 43-323, 46-323, 57-323,61-323, 64-323, 65-323, 68-323, 71-323, 72-323, 76-323, 79-323, 85-323,90-323, 94-323, 115-323, 118-323, 139-323, 150-323, 154-323, 169-323,172-323, 175-323, 178-323, 181-323, 183-323, 191-323, 192-323, 217-323,242-323, 242-257, 242-307, 246-257, 246-307, or 294-307 of SEQ ID NO:10.

In yet more preferred embodiments, GMG-3 or GMG-4 polypeptide fragmentshaving activity are selected from amino acids 20-333, 109-333, 125-333,128-333, 140-333, 160-333, 164-333, 179-333, 182-333, 185-333, 188-333,191-333, 193-333, 201-333, 202-333, 227-333, 252-333, 252-267, 252-317,256-267, 256-317, or 304-317 of SEQ ID NO: 2 or 4. In other yet morepreferred embodiments, Cluster 1 polypeptide fragments having activityare selected from amino acids 1-225, 17-225, 20-225, 32-225, 52-225,56-225, 71-225, 74-225, 77-225, 80-225, 83-225, 85-225, 93-225, 94-225,119-225, 144-225, 144-159, 144-209, 148-159, 148-209, or 196-209 of SEQID NO: 6. In other yet more preferred embodiments, GMG-6A polypeptidefragments having activity are selected from amino acids 20-330, 75-330,122-330, 125-330, 146-330, 157-330, 161-330, 176-330, 179-330, 182-330,185-330, 188-330, 190-330, 198-330, 199-330, 224-330, 249-330, 249-264,249-314, 253-264, 253-314 or 301-314 of SEQ ID NO: 8. In other yet morepreferred embodiments, GMG-6B polypeptide fragments having activity areselected from amino acids 20-323, 68-323, 115-323, 118-323, 139-323,150-323, 154-323, 169-323, 172-323, 175-323, 178-323, 181-323, 183-323,191-323, 192-323, 217-323, 242-323, 242-257, 242-307, 246-257, 246-307,or 294-307 of SEQ ID NO: 10.

In further preferred embodiments, said polypeptide fragment comprises anamino acid sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to the correspondingconsecutive amino acids of the polypeptide sequences identified in SEQID NO: 2, 4, 6, 8, or 10.

The invention further provides a purified or isolated polypeptidecomprising, consisting of, or consisting essentially of an amino acidsequence selected from the group consisting of: (a) a full-length atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical tothe corresponding amino acids polypeptide of SEQ ID NOs:2, 4, 6, 8, or10; (b) a full-length GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bpolypeptide of SEQ ID NOs:2, 4, 6, 8, or 10 absent the N-terminal Met;(c) a mature GMG-3, GMG-4, GMG-6A, or GMG-6B polypeptide of SEQ IDNOs:2, 4, 8, or 10 lacking signal peptide; (d) a GMG-3 or GMG-4polypeptide of SEQ ID NO:2 or 4 wherein said GMG-3 or GMG-4 polypeptideis of any one integer in length between 6 amino acids and 333 aminoacids (full-length) inclusive of SEQ ID NO:2 or 4, a Cluster 1polypeptide of SEQ ID NO: 6 wherein said Cluster 1 polypeptide is of anyone integer in length between 6 amino acids and 225 amino acids(full-length) inclusive of SEQ ID NO: 6, a GMG-6A polypeptide of SEQ IDNO: 8 wherein said GMG-6A polypeptide is of any one integer in lengthbetween 6 amino acids and 330 amino acids (full-length) inclusive of SEQID NO: 8, or a GMG-6B polypeptide of SEQ ID NO: 10 wherein said GMG-6Bpolypeptide is of any one integer in length between 6 amino acids and323 amino acids (full-length) inclusive of SEQ ID NO: 10; (e) theepitope-bearing fragments of a GMG-3, GMG-4, Cluster 1, GMG-6A, orGMG-6B polypeptide of SEQ ID NO: 2, 4, 6, 8, or 10; (f) a fragment of aGMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide of SEQ ID NOs: 2,4, 6, 8, or 10 comprising the globular head sequence TVFSRNVQVSLV (SEQID NO: 14 and having agonist activity, wherein said activity is selectedfrom the group consisting of lipid partitioning, lipid metabolism, andinsulin-like activity; (g) a fragment of a GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B polypeptide of SEQ ID NOs: 2, 4, 6, 8, or 10comprising the globular head sequence QVTGGERFNGLFAD (SEQ ID NO: 15) andhaving agonist activity, wherein said activity is selected from thegroup consisting of lipid partitioning, lipid metabolism, andinsulin-like activity; (h) the allelic variant polypeptides of any ofthe polypeptides of (a)-(g). The invention further provides forfragments of the polypeptides of(a)-(h) above, such as those havingbiological activity or comprising biologically functional domain(s).

In other highly preferred embodiments, GMG-3, GMG-4, Cluster 1, GMG-6A,or GMG-6B polypeptides comprise, consist essentially of, or consist of,a purified, isolated, or a recombinant GMG-3, GMG-4, Cluster 1, GMG-6A,or GMG-6B fragment comprised of all or part of the C-terminal globularC1q homology domain. Preferably, said GMG-3 or GMG-4 polypeptidefragment comprises, consists essentially of, or consists of, at least 6consecutive amino acids of amino acids 20-333 of SEQ ID NO: 2 or 4.Preferably, said Cluster 1 polypeptide fragment comprises, consistsessentially of, or consists of, at least 6 consecutive amino acids ofamino acids 1-225 of SEQ ID NO: 6. Preferably, said GMG-6A polypeptidefragment comprises, consists essentially of, or consists of, at least 6consecutive amino acids of amino acids 20-330 of SEQ ID NO: 8.Preferably, said GMG-6B polypeptide fragment comprises, consistsessentially of, or consists of, at least 6 consecutive amino acids ofamino acids 20-323 of SEQ ID NO: 10. In other preferred embodiments,said GMG-3 or GMG-4 polypeptide fragments having activity are selectedfrom amino acids 20-333, 43-333, 44-333, 45-333, 46-333, 47-333, 48-333,49-333, 50-333, 51-333, 52-333, 53-333, 54-333, 55-333, 56-333, 57-333,58-333, 59-333, 60-333, 61-333, 62-333, 63-333, 64-333, 65-333, 66-333,67-333, 68-333, 69-333, 70-333, 71-333, 72-333, 73-333, 74-333, 75-333,76-333, 77-333, 78-333, 79-333, 80-333, 81-333, 82-333, 83-333, 84-333,85-333, 86-333, 87-333, 88-333, 89-333, 90-333, 91-333, 92-333, 93-333,94-333, 95-333, 96-333, 97-333, 98-333, 99-333, 100-333, 101-333,102-333, 103-333, 104-333, 105-333, 106-333, 107-333, 108-333, 109-333,110-333, 111-333, 112-333, 113-333, 114-333, 115-333, 116-333, 117-333,118-333, 119-333, 120-333, 121-333, 122-333, 123-333, 124-333, 125-333,126-333, 127-333, 128-333, 129-333, 130-333, 131-333, 132-333, 133-333,134-333, 135-333, 136-333, 137-333, 138-333, 139-333, 140-333, 141-333,142-333, 143-333, 144-333, 145-333, 146-333, 147-333, 148-333, 149-333,150-333, 151-333, 152-333, 153-333, 154-333, 155-333, 156-333, 157-333,158-333, 159-333, 160-333, 161-333, 162-333, 163-333, 164-333, 165-333,166-333, 167-333, 168-333, 169-333, 170-333, 171-333, 172-333, 173-333,174-333, 175-333, 176-333, 177-333, 178-333, 179-333, 180-333, 181-333,182-333, 183-333, 184-333, 185-333, 186-333, 187-333, 188-333, 189-333,190-333, 191-333, 192-333, 193-333, 194-333, 195-333, 196-333, 197-333,198-333, 199-333, 200-333, 201-333 or 202-333 of SEQ ID NO: 2 or 4. Inother preferred embodiments, said Cluster 1 polypeptide fragments havingactivity are selected from amino acids 1-225, 2-225, 3-225, 4-225,5-225, 6-225, 7-225, 8-225, 9-225, 10-225, 11-225, 12-225, 13-225,14-225, 15-225, 16-225, 17-225, 18-225, 19-225, 20-225, 21-225, 22-225,23-225, 24-225, 25-225, 26-225, 27-225, 28-225, 29-225, 30-225, 31-225,32-225, 33-225, 34-225, 35-225, 36-225, 37-225, 38-225, 39-225, 40-225,41-225, 42-225, 43-225, 44-225, 45-225, 46-225, 47-225, 48-225, 49-225,50-225, 51-225, 52-225, 53-225, 54-225, 55-225, 56-225, 57-225, 58-225,59-225, 60-225, 61-225, 62-225, 63-225, 64-225, 65-225, 66-225, 67-225,68-225, 69-225, 70-225, 71-225, 72-225, 73-225, 74-225, 75-225, 76-225,77-225, 78-225, 79-225, 80-225, 81-225, 82-225, 83-225, 84-225, 85-225,86-225, 87-225, 88-225, 89-225, 90-225, 91-225, 92-225, 93-225 or 94-225of SEQ ID NO: 6. In other preferred embodiments, said GMG-6A polypeptidefragments having activity are selected from amino acids 20-330, 43-330,44-330, 45-330, 46-330, 47-330, 48-330, 49-330, 50-330, 51-330, 52-330,53-330, 54-330, 55-330, 56-330, 57-330, 58-330, 59-330, 60-330, 61-330,62-330, 63-330, 64-330, 65-330, 66-330, 67-330, 68-330, 69-330, 70-330,71-330, 72-330, 73-330, 74-330, 75-330, 76-330, 77-330, 78-330, 79-330,80-330, 81-330, 82-330, 83-330, 84-330, 85-330, 86-330, 87-330, 88-330,89-330, 90-330, 91-330, 92-330, 93-330, 94-330, 95-330, 96-330, 97-330,98-330, 99-330, 100-330, 101-330, 102-330, 103-330, 104-330, 105-330,106-330, 107-330, 108-330, 109-330, 110-330, 111-330, 112-330, 113-330,114-330, 115-330, 116-330, 117-330, 118-330, 119-330, 120-330, 121-330,122-330, 123-330, 124-330, 125-330, 126-330, 127-330, 128-330, 129-330,130-330, 131-330, 132-330, 133-330, 134-330, 135-330, 136-330, 137-330,138-330, 139-330, 140-330, 141-330, 142-330, 143-330, 144-330, 145-330,146-330, 147-330, 148-330, 149-330, 150-330, 151-330, 152-330, 153-330,154-330, 155-330, 156-330, 157-330, 158-330, 159-330, 160-330, 161-330,162-330, 163-330, 164-330, 165-330, 166-330, 167-330, 168-330, 169-330,170-330, 171-330, 172-330, 173-330, 174-330, 175-330, 176-330, 177-330,178-330, 179-330, 180-330, 181-330, 182-330, 183-330, 184-330, 185-330,186-330, 187-330, 188-330, 189-330, 190-330, 191-330, 192-330, 193-330,194-330, 195-330, 196-330, 197-330, 198-330 or 199-330 of SEQ ID NO: 8.In other preferred embodiments, said GMG-6B polypeptide fragments havingactivity are selected from amino acids 20-323, 43-323, 44-323, 45-323,46-323, 47-323, 48-323, 49-323, 50-323, 51-323, 52-323, 53-323, 54-323,55-323, 56-323, 57-323, 58-323, 59-323, 60-323, 61-323, 62-323, 63-323,64-323, 65-323, 66-323, 67-323, 68-323, 69-323, 70-323, 71-323, 72-323,73-323, 74-323, 75-323, 76-323, 77-323, 78-323, 79-323, 80-323, 81-323,82-323, 83-323, 84-323, 85-323, 86-323, 87-323, 88-323, 89-323, 90-323,91-323, 92-323, 93-323, 94-323, 95-323, 96-323, 97-323, 98-323, 99-323,100-323, 101-323, 102-323, 103-323, 104-323, 105-323, 106-323, 107-323,108-323, 109-323, 110-323, 111-323, 112-323, 113-323, 114-323, 115-323,116-323, 117-323, 118-323, 119-323, 120-323, 121-323, 122-323, 123-323,124-323, 125-323, 126-323, 127-323, 128-323, 129-323, 130-323, 131-323,132-323, 133-323, 134-323, 135-323, 136-323, 137-323, 138-323, 139-323,140-323, 141-323, 142-323, 143-323, 144-323, 145-323, 146-323, 147-323,148-323, 149-323, 150-323, 151-323, 152-323, 153-323, 154-323, 155-323,156-323, 157-323, 158-323, 159-323, 160-323, 161-323, 162-323, 163-323,164-323, 165-323, 166-323, 167-323, 168-323, 169-323, 170-323, 171-323,172-323, 173-323, 174-323, 175-323, 176-323, 177-323, 178-323, 179-323,180-323, 181-323, 182-323, 183-323, 184-323, 185-323, 186-323, 187-323,188-323, 189-323, 190-323, 191-323 or 192-323 of SEQ ID NO: 10.

In more preferred embodiments, said GMG-3 or GMG-4 polypeptide fragmentscomprised of all or part of the C-terminal globular C1q homology domainand having activity are selected from amino acids 20-333, 43-333,45-333, 46-333, 50-333, 53-333, 61-333, 67-333, 74-333, 75-333, 77-333,81-333, 82-333, 86-333, 89-333, 95-333, 100-333, 104-333, 109-333,113-333, 116-333, 125-333, 128-333, 140-333, 160-333, 164-333, 179-333,182-333, 185-333, 188-333, 191-333, 193-333, 201-333, 202-333, 227-333,252-333, 252-267, 252-317, 256-267, 256-317, or 304-317 of SEQ ID NO: 2or 4. In other more preferred embodiments, said Cluster 1 polypeptidefragments having activity are selected from amino acids 1-225, 5-225,8-225, 17-225, 20-225, 32-225, 52-225, 56-225, 71-225, 74-225, 77-225,80-225, 83-225, 85-225, 93-225, 94-225, 119-225, 144-225, 144-159,144-209, 148-159, 148-209, or 196-209 of SEQ ID NO: 6. In other morepreferred embodiments, said GMG-6A polypeptide fragments having activityare selected from amino acids 20-330, 43-330, 45-330, 46-330, 50-333,53-330, 64-330, 68-330, 71-330, 72-330, 75-330, 78-330, 79-330, 83-330,86-330, 92-330, 97-330, 101-330, 122-330, 125-330, 146-330, 157-330,161-330, 176-330, 179-330, 182-330, 185-330, 188-330, 190-330, 198-330,199-330, 224-330, 249-330, 249-264, 249-314, 253-264, 253-314 or 301-314of SEQ ID NO: 8. In other more preferred embodiments, said GMG-6Bpolypeptide fragments having activity are selected from amino acids20-323, 43-323, 46-323, 57-323, 61-323, 64-323, 65-323, 68-323, 71-323,72-323, 76-323, 79-323, 85-323, 90-323, 94-323, 115-323, 118-323,139-323, 150-323, 154-323, 169-323, 172-323, 175-323, 178-323, 181-323,183-323, 191-323, 192-323, 217-323, 242-323, 242-257, 242-307, 246-257,246-307, or 294-307 of SEQ ID NO: 10.

In yet more preferred embodiments, said GMG-3 or GMG-4 polypeptidefragments comprised of all or part of the C-terminal globular C1qhomology domain and having activity are selected from amino acids20-333, 109-333, 125-333, 128-333, 140-333, 160-333, 164-333, 179-333,182-333, 185-333, 188-333, 191-333, 193-333, 201-333, 202-333, 227-333,252-333, 252-267, 252-317, 256-267, 256-317, or 304-317 of SEQ ID NO: 2or 4. In other yet more preferred embodiments, said Cluster 1polypeptide fragments having activity are selected from amino acids1-225, 17-225, 20-225, 32-225, 52-225, 56-225, 71-225, 74-225, 77-225,80-225, 83-225, 85-225, 93-225, 94-225, 119-225, 144-225, 144-159,144-209, 148-159, 148-209, or 196-209 of SEQ ID NO: 6. In other yet morepreferred embodiments, said GMG-6A polypeptide fragments having activityare selected from amino acids 20-330, 75-330, 122-330, 125-330, 146-330,157-330, 161-330, 176-330, 179-330, 182-330, 185-330, 188-330, 190-330,198-330, 199-330, 224-330, 249-330, 249-264, 249-314, 253-264, 253-314or 301-314 of SEQ ID NO: 8. In other yet more preferred embodiments,said GMG-6B polypeptide fragments having activity are selected fromamino acids 20-323, 68-323, 115-323, 118-323, 139-323, 150-323, 154-323,169-323, 172-323, 175-323, 178-323, 181-323, 183-323, 191-323, 192-323,217-323, 242-323, 242-257, 242-307, 246-257, 246-307, or 294-307 of SEQID NO: 10.

Alternatively, said GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bpolypeptide fragment comprises, consists essentially of, or consists of,an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to the corresponding amino acids 197-333 of SEQ IDNO: 2 or 4, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the corresponding amino acids 88-225 of SEQ ID NO: 6, atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical tothe corresponding amino acids 197-330 of SEQ ID NO: 8, or at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to thecorresponding amino acids 190-323 of SEQ ID NO: 10.

In a further preferred embodiment, GMG-3, GMG-4, Cluster 1, GMG-6A, andGMG-6B polypeptides are able to lower circulating (either in blood,serum or plasma) levels (concentration) of: (i) free fatty acids, (ii)glucose, and/or (iii) triglycerides. Further preferred polypeptides ofthe invention demonstrating free fatty acid level lowering activity,glucose level lowering activity, and/or triglyceride level loweringactivity, have an activity that is the same or greater than full-lengthGMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides at the samemolar concentration, have the same or greater than transient activityand/or have a sustained activity.

Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6Bpolypeptides are those that significantly stimulate muscle lipid or freefatty acid oxidation. Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A,and GMG-6B polypeptides are those that significantly stimulate musclelipid or free fatty acid oxidation.

Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6Bpolypeptides are those that cause C2C12 cells differentiated in thepresence of said polypeptides to undergo at least 10%, 20%, 30%, 35%, or40% more oleate oxidation as compared to untreated cells.

Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6Bpolypeptides are those that increase leptin uptake in a liver cell line(preferably BPRCL mouse liver cells (ATCC CRL-2217)).

Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6Bpolypeptides are those that significantly reduce the postprandialincrease in plasma free fatty acids due to a high fat meal.

Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6Bpolypeptides are those that significantly reduce or eliminate ketonebody production as the result of a high fat meal.

Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6Bpolypeptides are those that increase glucose uptake in skeletal musclecells.

Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6Bpolypeptides are those that increase glucose uptake in adipose cells.

Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6Bpolypeptides are those that increase glucose uptake in neuronal cells.

Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6Bpolypeptides are those that increase glucose uptake in red blood cells.

Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6Bpolypeptides are those that increase glucose uptake in the brain.

Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6Bpolypeptides are those that significantly reduce the postprandialincrease in plasma glucose following a meal, particularly a highcarbohydrate meal.

Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6Bpolypeptides are those that significantly prevent the postprandialincrease in plasma glucose following a meal, particularly a high fat ora high carbohydrate meal.

Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6Bpolypeptides are those that increase insulin sensitivity.

Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6Bpolypeptides are those that inhibit the progression from impairedglucose tolerance to insulin resistance.

Further preferred GMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6Bpolypeptides are those that form multimers (e.g., heteromultimers orhomomultimers) in vitro and/or in vivo. Preferred multimers arehomodimers or homotrimers. Other preferred multimers are homomultimerscomprising at least 4, 6, 8, 9, 10 or 12 GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B polypeptide subunits. Other preferred mulimers arehetero multimers comprising a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bpolypeptide of the invention.

Further preferred embodiments include heterologous polypeptidescomprising one of the GMG 3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bpolypeptides of the invention.

In a second aspect, the invention features purified, isolated, orrecombinant polynucleotides encoding said GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B polypeptides described in the first aspect, or thecomplement thereof. A further preferred embodiment of the invention is arecombinant, purified or isolated polynucleotide comprising, orconsisting of a mammalian genomic sequence, gene, or fragments thereof.In one aspect the sequence is derived from a human, mouse or othermammal. In a preferred aspect, the genomic sequence includes isolated,purified, or recombinant polynucleotides comprising a contiguous span ofat least 12, 15, 18, 20, 22, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100,150, 200, 500, 1000, 2000, 5000, 10000 or 50000 nucleotides of any oneof the polynucleotide sequences described in SEQ ID NOs: 1, 3, 5, 7, or9, or the complements thereof, wherein said contiguous span comprises anucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to the corresponding nucleotide sequence of theC-terminal globular C1q homology domains of SEQ ID NOs: 1, 3, 5, 7, or9. In further embodiments the polynucleotides are DNA, RNA, DNA/RNAhybrids, single-stranded, and double-stranded.

In a third aspect, the invention features a recombinant vectorcomprising, consisting essentially of, or consisting of, saidpolynucleotide described in the second aspect.

In a fourth aspect, the invention features a recombinant cellcomprising, consisting essentially of, or consisting of, saidrecombinant vector described in the third aspect. A further embodimentincludes a host cell recombinant for a polynucleotide of the invention.

In a fifth aspect, the invention features a pharmaceutical orphysiologically acceptable composition comprising, consistingessentially of, or consisting of, said GMG-3, GMG-4, Cluster 1, GMG-6A,or GMG-6B polypeptides described in the first aspect and, alternatively,a pharmaceutical or physiologically acceptable diluent.

In a sixth aspect, the invention features a method of reducing body masscomprising providing or administering to individuals in need of reducingbody mass said pharmaceutical or physiologically acceptable compositiondescribed in the fifth aspect.

In preferred embodiments, the identification of said individuals in needof reducing body mass to be treated with said pharmaceutical orphysiologically acceptable composition comprises genotyping GMG-3,GMG-4, Cluster 1, GMG-6A, or GMG-6B single nucleotide polymorphisms(SNPs) or measuring metabolic polypeptide or mRNA levels in clinicalsamples from said individuals. Preferably, said clinical samples areselected from the group consisting of plasma, urine, and saliva.Preferably, a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptidefragment of the present invention is administered to an individual withat least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%reduction in blood, serum or plasma levels of full-length any one or allof the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides or thenaturally proteolytically cleaved GMG-3, GMG-4, Cluster 1, GMG-6A, orGMG-6B fragments as compared to healthy, non-obese patients.

In a seventh aspect, the invention features a method of preventing ortreating an metabolic-related disease or disorder comprising providingor administering to an individual in need of such treatment saidpharmaceutical or physiologically acceptable composition described inthe fifth aspect. In preferred embodiments, the identification of saidindividuals in need of such treatment to be treated with saidpharmaceutical or physiologically acceptable composition comprisesgenotyping GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B single nucleotidepolymorphisms (SNPs) or measuring GMG-3, GMG-4, Cluster 1, GMG-6A, orGMG-6B polypeptide or mRNA levels in clinical samples from saidindividuals. Preferably, said clinical samples are selected from thegroup consisting of blood, serum, plasma, urine, and saliva. Preferably,said metabolic-related disease or disorder is selected from the groupconsisting of obesity, impaired glucose tolerance, insulin resistance,atherosclerosis, atheromatous disease, heart disease, hypertension,stroke, Syndrome X, non-insulin-dependent diabetes and Type II diabetes.Type II diabetes-related complications to be treated by the methods ofthe invention include microangiopathic lesions, ocular lesions, andrenal lesions. Heart disease includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure. Othermetabolic-related disorders to be treated by compounds of the inventioninclude hyperlipidemia and hyperuricemia. Yet other metabolic-relateddiseases or disorders of the invention include cachexia, wasting,AIDS-related weight loss, cancer-related weight loss, anorexia, andbulimia. In preferred embodiments, said individual is a mammal,preferably a human.

In related aspects, embodiments of the present invention includesmethods of causing or inducing a desired biological response in anindividual comprising the steps of: providing or administering to anindividual a composition comprising a GMG-3, GMG-4, Cluster 1, GMG-6A,or GMG-6B polypeptide, wherein said biological response is selected fromthe group consisting of:

(a) modulating circulating (either blood, serum, or plasma) levels(concentration) of free fatty acids, wherein said modulating ispreferably lowering;

(b) modulating circulating (either blood, serum or plasma) levels(concentration) of glucose, wherein said modulating is preferablylowering;

(c) modulating circulating (either blood, serum or plasma) levels(concentration) of triglycerides, wherein said modulating is preferablylowering;

(d) stimulating muscle lipid or free fatty acid oxidation;

(c) modulating leptin uptake in the liver or liver cells, wherein saidmodulating is preferably increasing;

(e) modulating the postprandial increase in plasma free fatty acids dueto a high fat meal, wherein said modulating is preferably reducing;

(f) modulating ketone body production as the result of a high fat meal,wherein said modulating is preferably reducing or eliminating;

(g) increasing cell or tissue sensitivity to insulin, particularlymuscle, adipose, liver or brain; and

(h) inhibiting the progression from impaired glucose tolerance toinsulin resistance;

and further wherein said biological response is significantly greaterthan, or at least 10%, 20%, 30%, 35%, 40%, 50% 75% 100% or 500% greaterthan, the biological response caused or induced by insulin alone at thesame molar concentration. In further preferred embodiments, the presentinvention of said pharmaceutical or physiologically acceptablecomposition can be used as a method to control blood glucose in somepersons with Non-Insulin Dependent Diabetes Mellitus (NIDDM, Type IIdiabetes) in combination with insulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to control blood glucose in some persons with Insulin DependentDiabetes Mellitus (IDDM, Type I diabetes) in combination with insulintherapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to control body weight in some persons with Non-InsulinDependent Diabetes Mellitus (NIDDM, Type II diabetes) in combinationwith insulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to control body weight in some persons with Insulin DependentDiabetes Mellitus (IDDM, Type I diabetes) in combination with insulintherapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to control blood glucose in some persons with Non-InsulinDependent Diabetes Mellitus (NIDDM, Type II diabetes) alone, withoutcombination of insulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to control blood glucose in some persons with Insulin DependentDiabetes Mellitus (IDDM, Type I diabetes) alone, without combination ofinsulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to control body weight in some persons with Non-InsulinDependent Diabetes Mellitus (NIDDM, Type II diabetes) alone, withoutcombination of insulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to control body weight in some persons with Insulin DependentDiabetes Mellitus (IDDM, Type I diabetes) alone, without combination ofinsulin therapy.

In a further preferred embodiment, the present invention may be used incomplementary therapy of NIDDM patients to improve their weight orglucose control in combination with an insulin secretagogue (preferablyoral form) or an insulin sensitising (preferably oral form) agent.Preferably, the oral insulin secretagogue is 1,1-dimethyl-2-(2-morpholino phenyl)guanidine fumarate (BTS67582) or asulphonylurea selected from tolbutamide, tolazamide, chlorpropamide,glibenclamide, glimepiride, glipizide and glidazide. Preferably, theinsulin sensitising agent is selected from metformin, ciglitazone,troglitazone and pioglitazone.

The present invention further provides a method of improving the bodyweight or glucose control of NIDDM patients alone, without an insulinsecretagogue or an insulin sensitising agent.

In a further preferred embodiment, the present invention may be used incomplementary therapy of IDDM patients to improve their weight orglucose control in combination with an insulin secretagogue (preferablyoral form) or an insulin sensitising (preferably oral form) agent.Preferably, the insulin secretagogue is 1,1-dimethyl-2-(2-morpholinophenyl)guanidine fumarate (BTS67582) or a sulphonylurea selected fromtolbutamide, tolazamide, chlorpropamide, glibenclamide, glimepiride,glipizide and glidazide. Preferably, the insulin sensitising agent isselected from metformin, ciglitazone, troglitazone and pioglitazone.

The present invention further provides a method of improving the bodyweight or glucose control of IDDM patients alone, without an insulinsecretagogue or an insulin sensitising agent.

In a further preferred embodiment, the present invention may beadministered either concomitantly or concurrently, with the insulinsecretagogue or insulin sensitising agent for example in the form ofseparate dosage units to be used simultaneously, separately orsequentially (either before or after the secretagogue or either beforeor after the sensitising agent). Accordingly, the present inventionfurther provides for a composition of pharmaceutical or physiologicallyacceptable composition and an insulin secretagogue or insulinsensitising agent as a combined preparation for simultaneous, separateor sequential use for the improvement of body weight or glucose controlin NIDDM or IDDM patients.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition furtherprovides a method for the use as an insulin sensitiser.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to improve insulin sensitivity in some persons with Non-InsulinDependent Diabetes Mellitus (NIDDM, Type II diabetes) in combinationwith insulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to improve insulin sensitivity in some persons with InsulinDependent Diabetes Mellitus (IDDM, Type I diabetes) in combination withinsulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to improve insulin sensitivity in some persons with Non-InsulinDependent Diabetes Mellitus (NIDDM, Type II diabetes) without insulintherapy.

In an eighth aspect, the invention features a method of making theGMG-3, GMG-4, Cluster 1, GMG-6A, and GMG-6B polypeptides described inthe first aspect, wherein said method is selected from the groupconsisting of: proteolytic cleavage, recombinant methodology andartificial synthesis.

In a ninth aspect, the present invention provides a method of making arecombinant GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptidefragment or a full length GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bpolypeptide, the method comprising providing a transgenic, non-humanmammal whose milk contains said recombinant GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B polypeptide fragment or full-length protein, andpurifying said recombinant GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bpolypeptide fragment or said full-length GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B polypeptide from the milk of said non-human mammal. Inone embodiment, said non-human mammal is a cow, goat, sheep, rabbit, ormouse. In another embodiment, the method comprises purifying arecombinant full-length GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bpolypeptide from said milk, and further comprises cleaving said proteinin vitro to obtain a desired GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bpolypeptide fragment.

In a tenth aspect, the invention features a purified or isolatedantibody capable of specifically binding to a polypeptide of the presentinvention. In one aspect of this embodiment, the antibody is capable ofbinding to a polypeptide comprising at least 6 consecutive amino acids,at least 8 consecutive amino acids, or at least 10 consecutive aminoacids of the sequence of one of the polypeptide sequences described inSEQ ID NOs: 2, 4, 6, 8, or 10.

In an eleventh aspect, the invention features a use of the polypeptidedescribed in the first aspect for treatment of metabolic-relateddiseases and disorders and/or reducing or increasing body mass.Preferably, said metabolic-related diseases and disorders are selectedfrom the group consisting of obesity, insulin resistance,atherosclerosis, atheromatous disease, heart disease, hypertension,stroke, Syndrome X, non-insulin-dependent diabetes and Type II diabetes.Type II diabetes-related complications to be treated by the methods ofthe invention include microangiopathic lesions, ocular lesions, andrenal lesions. Heart disease includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure. Othermetabolic-related disorders to be treated by compounds of the inventioninclude hyperlipidemia and hyperuricemia. Yet other metabolic-relateddiseases or disorders of the invention include cachexia, wasting,AIDS-related weight loss, anorexia, and bulimia. In preferredembodiments, said individual is a mammal, preferably a human.

In a twelfth aspect, the invention provides a polypeptide of the firstaspect of the invention, or a composition of the fifth aspect of theinvention, for use in a method of treatment of the human or animal body.

In a thirteenth aspect, the invention features methods of reducing bodyweight for cosmetic purposes comprising providing to an individual saidpharmaceutical or physiologically acceptable composition described inthe fifth aspect, or a polypeptide described in the first aspect.Preferably, for said reducing body weight said individual has a BMI ofat least 20 and no more than 25. Alternatively, for said increasing bodyweight said individual preferably has a BMI of at least 15 and no morethan 20.

In a fourteenth aspect, the invention features the pharmaceutical orphysiologically acceptable composition described in the fifth aspect forreducing body mass and/or for treatment or prevention ofmetabolic-related diseases or disorders. Preferably, saidmetabolic-related disease or disorder is selected from the groupconsisting of obesity, impaired glucose tolerance, insulin resistance,atherosclerosis, atheromatous disease, heart disease, hypertension,stroke, Syndrome X, non-insulin-dependent diabetes and Type II diabetes.Type II diabetes-related complications to be treated by the methods ofthe invention include microangiopathic lesions, ocular lesions, andrenal lesions. Heart disease includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure. Othermetabolic-related disorders to be treated by compounds of the inventioninclude hyperlipidemia and hyperuricemia. Yet other metabolic-relateddiseases or disorders of the invention include cachexia, wasting,ADS-related weight loss, cancer-related weight loss, anorexia, andbulimia. In preferred embodiments, said individual is a mammal,preferably a human. In preferred embodiments, the identification of saidindividuals to be treated with said pharmaceutical or physiologicallyacceptable composition comprises genotyping GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B single nucleotide polymorphisms (SNPs) or measuringGMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides or mRNA levelsin clinical samples from said individuals. Preferably, said clinicalsamples are selected from the group consisting of blood, serum, plasma,urine, and saliva.

In a fifteenth aspect, the invention features the pharmaceutical orphysiologically acceptable composition described in the fifth aspect forreducing body weight for cosmetic reasons.

In a sixteenth aspect, the invention features methods of treatinginsulin resistance comprising providing to an individual saidpharmaceutical or physiologically acceptable composition described inthe fifth aspect, or a polypeptide described in the first aspect.

In a seventeenth aspect, the invention features the pharmaceutical orphysiologically acceptable composition described in the fifth aspect ina method of treating individuals with normal glucose tolerance (NGT) whoare obese or who have fasting hyperinsulinemia, or who have both.

In further preferred embodiments, the invention features thepharmaceutical or physiologically acceptable composition described inthe fifth aspect in a method of treating individuals with gestationaldiabetes. Gestational diabetes refers to the development of diabetes inan individual during pregnancy, usually during the second or thirdtrimester of pregnancy.

In further preferred embodiments, the invention features thepharmaceutical or physiologically acceptable composition described inthe fifth aspect in a method of treating individuals with impairedfasting glucose (IFG). Impaired fasting glucose (IFG) is that conditionin which fasting plasma glucose levels in an individual are elevated butnot diagnostic of overt diabetes, i.e. plasma glucose levels of lessthan 126 mg/dl and less than or equal to 110 mg/dl.

In further preferred embodiments, the invention features thepharmaceutical or physiologically acceptable composition described inthe fifth aspect in a method of treating and preventing impaired glucosetolerance (IGT) in an individual. By providing therapeutics and methodsfor reducing or preventing IGT, i.e., for normalizing insulinresistance, the progression to NIDDM can be delayed or prevented.Furthermore, by providing therapeutics and methods for reducing orpreventing insulin resistance, the invention provides methods forreducing and/or preventing the appearance of Insulin-ResistanceSyndrome.

In further preferred embodiments, the invention features thepharmaceutical or physiologically acceptable composition described inthe fifth aspect in a method of treating a subject having polycysticovary syndrome (PCOS). PCOS is among the most common disorders ofpremenopausal women, affecting 5-10% of this population.Insulin-sensitizing agents, e.g., troglitazone, have been shown to beeffective in PCOS and that, in particular, the defects in insulinaction, insulin secretion, ovarian steroidogenosis and fibrinolysis areimproved (Ehrman et al. (1997) J Clin Invest 100:1230), such as ininsulin-resistant humans. Accordingly, the invention provides methodsfor reducing insulin resistance, normalizing blood glucose thus treatingand/or preventing PCOS.

In further preferred embodiments, the invention features thepharmaceutical or physiologically acceptable composition described inthe fifth aspect in a method of treating a subject having insulinresistance.

In further preferred embodiments, a subject having insulin resistance istreated according to the methods of the invention to reduce or cure theinsulin-resistance. As insulin resistance is also often associated withinfections and cancer, prevention or reducing insulin resistanceaccording to the methods of the invention may prevent or reduceinfections and cancer.

In further preferred embodiment, the methods of the invention are usedto prevent the development of insulin resistance in a subject, e.g.,those known to have an increased risk of developing insulin-resistance.

Thus, any of the above-described tests or other tests known in the artcan be used to determine that a subject is insulin-resistant, whichpatient can then be treated according to the methods of the invention toreduce or cure the insulin-resistance. Alternatively, the methods of theinvention can also be used to prevent the development of insulinresistance in a subject, e.g., those known to have an increased risk ofdeveloping insulin-resistance.

In an eighteenth aspect, the invention features a method of preventingor treating an metabolic-related disease or disorder comprisingproviding or administering to an individual in need of such treatmentsaid pharmaceutical or physiologically acceptable composition describedin the fifth aspect. In preferred embodiments, the identification ofsaid individuals in need of such treatment to be treated with saidpharmaceutical or physiologically acceptable composition comprisesgenotyping GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B single nucleotidepolymorphisms (SNPs) or measuring GMG-3, GMG-4, Cluster 1, GMG-6A, orGMG-6B polypeptide or mRNA levels in clinical samples from saidindividuals. Preferably, said clinical samples are selected from thegroup consisting of blood, serum, plasma, urine, and saliva. Preferably,said metabolic-related disease or disorder is selected from the groupconsisting of obesity, impaired glucose tolerance, insulin resistance,atherosclerosis, atheromatous disease, heart disease, hypertension,stroke, Syndrome X, non-insulin-dependent diabetes and Type II diabetes.Type II diabetes-related complications to be treated by the methods ofthe invention include microangiopathic lesions, ocular lesions, andrenal lesions. Heart disease includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure. Othermetabolic-related disorders to be treated by compounds of the inventioninclude hyperlipidemia and hyperuricemia. Yet other metabolic-relateddiseases or disorders of the invention include cachexia, wasting,FIV-related weight loss, cancer-related weight loss, anorexia, andbulimia. In preferred embodiments, said individual is a mammal,preferably non-human, preferably a cat or a dog.

In a nineteenth aspect, the invention features a method of using aGMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide or polypeptidefragment to screen compounds for one or more antagonists of GMG-3,GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide or polypeptide fragmentactivity, wherein said activity is selected from but not restricted tolipid partitioning, lipid metabolism, and insulin-like activity.

In preferred embodiment, said compound is selected from but is notrestricted to small molecular weight organic or inorganic compound,protein, peptide, carbohydrate, or lipid.

In a twentieth aspect, the invention features a method of using a GMG-3,GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide to identify one or morecell types expressing a cell surface receptor for said GMG-3, GMG-4,Cluster 1, GMG-6A, or GMG-6B polypeptide, preferably wherein saidpolypeptide comprises all or part of the C-terminal globular C1qhomology domain and has lipid partitioning, lipid metabolism, orinsulin-like activities.

In a twenty-first aspect, the invention features a method of using aGMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide to clone cDNAencoding a cell surface receptor for said GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B polypeptide, preferably wherein said polypeptidecomprises all or part of the C-terminal globular C1q homology domain andhas lipid partitioning, lipid metabolism, or insulin-like activities.

In a twenty-second aspect, the invention features a method of using aGMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polynucleotide to generatetransgenic non-human mammals expressing GMG-3, GMG-4, Cluster 1, GMG-6A,or GMG-6B polypeptides, preferably wherein said non-human mammal ismouse, cow, sheep, goat, pig, or rabbit.

In a twenty-third aspect, the invention features a method of using agenomic polynucleotide or fragment thereof of SEQ ID NO: 11, 12, or 13to generate a transgenic mouse in which expression of the gene encodingGMG-6A and GMG-6B is knocked-out either globally or in a tissue-specificmanner.

In a preferred aspect of the methods above and disclosed herein, theamount of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide orpolynucleotide administered to an individual is sufficient to bringcirculating (blood, serum, or plasma) levels (concentration) of GMG-3,GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides to their normal levels(levels in non-obese individuals). “Normal levels” may be specified asthe total concentration of all circulating GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B polypeptides (full-length GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B proteins and fragments thereof) or the concentrationof all circulating proteolytically cleaved GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B polypeptides only.

In a further preferred aspect of the methods above and disclosed herein,weight loss is due in part or in whole to a decrease in mass of eithera) subcutaneous adipose tissue and/or b) visceral (omental) adiposetissue.

Full-length GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides andpolynucleotides encoding the same may be specifically substituted for aGMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide fragment orpolynucleotide encoding the same in any embodiment of the presentinvention.

It is further understood that by GMG-3 polypeptide is meant the aminoacid sequence of SEQ ID NO: 2 as well as any related polypeptideincorporating one or more of the amino acid polymorphisms indicated inthe sequence listing, namely the related polypeptide with Val atposition 219, the related polypeptide with Met at position 301, and therelated polypeptide with Val at position 219 and Met at position 301.

It is further understood that by GMG-4 polypeptide is meant the aminoacid sequence of SEQ ID NO: 4 as well as any related polypeptideincorporating one or more of the amino acid polymorphisms indicated inthe sequence listing, namely the related polypeptide with Ala atposition 238.

It is further understood that by Cluster 1 polypeptide is meant theamino acid sequence of SEQ ID NO: 6 as well as any related polypeptideincorporating one or more of the amino acid polymorphisms indicated inthe sequence listing, namely the related polypeptide with Ala atposition 130.

DETAILED DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO:1 represents the cDNA sequence of GMG-3.

SEQ ID NO:2 represents the amino acid sequence encoded by the cDNA ofSEQ ID NO:1.

SEQ ID NO:3 represents the cDNA sequence of GMG-4.

SEQ ID NO:4 represents the amino acid sequence encoded by the cDNA ofSEQ ID NO:3.

SEQ ID NO:5 represents the polynucleotide sequence of Cluster 1.

SEQ ID NO:6 represents the amino acid sequence encoded by thepolynucleotide sequence of SEQ ID NO:5.

SEQ ID NO:7 represents the cDNA sequence of GMG-6A.

SEQ ID NO:8 represents the amino acid sequence encoded by the cDNA ofSEQ ID NO:7.

SEQ ID NO:9 represents the cDNA sequence of GMG-6B.

SEQ ID NO:10 represents the amino acid sequence encoded by the cDNA ofSEQ ID NO:9.

SEQ ID NO:11 represents GMG-6 genomic sequence comprising the codingregion of the first coding exon for GMG-6A and GMG-6B.

SEQ ID NO:12 represents GMG-6 genomic sequence comprising the codingregion of the second coding exon for GMG-6A and GMG-6B.

SEQ ID NO:13 represents GMG-6 genomic sequence comprising the codingregion of the third coding exon for GMG-6A and GMG-6B.

SEQ ID NOs:14 and 15 are subsequences of SEQ ID NOs:2, 4, 6, 8, and 10.

DETAILED DESCRIPTION OF THE INVENTION

Before describing the invention in greater detail, the followingdefinitions are set forth to illustrate and define the meaning and scopeof the terms used to describe the invention herein.

As used interchangeably herein, the terms “oligonucleotides”, and“polynucleotides” and nucleic acid include RNA, DNA, or RNA/DNA hybridsequences of more than one nucleotide in either single chain or duplexform. The terms encompass “modified nucleotides” which comprise at leastone modification, including by way of example and not limitation: (a) analternative linking group, (b) an analogous form of purine, (c) ananalogous form of pyrimidine, or (d) an analogous sugar. For examples ofanalogous linking groups, purines, pyrimidines, and sugars see forexample PCT publication No. WO 95/04064. The polynucleotide sequences ofthe invention may be prepared by any known method, including synthetic,recombinant, ex vivo generation, or a combination thereof, as well asutilizing any purification methods known in the art.

The terms polynucleotide construct, recombinant polynucleotide andrecombinant polypeptide are used herein consistently with their use inthe art. The terms “upstream” and “downstream” are also used hereinconsistently with their use in the art. The terms “base paired” and“Watson & Crick base paired” are used interchangeably herein andconsistently with their use in the art. Similarly, the terms“complementary”, “complement thereof”, “complement”, “complementarypolynucleotide”, “complementary nucleic acid” and “complementarynucleotide sequence” are used interchangeably herein and consistentlywith their use in the art.

The term “purified” is used herein to describe a polynucleotide orpolynucleotide vector of the invention that has been separated fromother compounds including, but not limited to, other nucleic acids,carbohydrates, lipids and proteins (such as the enzymes used in thesynthesis of the polynucleotide). Purified can also refer to theseparation of covalently closed polynucleotides from linearpolynucleotides, or vice versa, for example. A polynucleotide issubstantially pure when at least about 50%, 60%, 75%, or 90% of a samplecontains a single polynucleotide sequence. In some cases this involves adetermination between conformations (linear versus covalently closed). Asubstantially pure polynucleotide typically comprises about 50, 60, 70,80, 90, 95, 99% weight/weight of a nucleic acid sample. Polynucleotidepurity or homogeneity may be indicated by a number of means well knownin the art, such as agarose or polyacrylamide gel electrophoresis of asample, followed by visualizing a single polynucleotide band uponstaining the gel. For certain purposes, higher resolution can beachieved by using HPLC or other means well known in the art.

Similarly, the term “purified” is used herein to describe a polypeptideof the invention that has been separated from other compounds including,but not limited to, nucleic acids, lipids, carbohydrates and otherproteins. In some preferred embodiments, a polypeptide is substantiallypure when at least about 50%, 60%, 75%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or 99.5% of the polypeptide molecules of a sample have a singleamino acid sequence. In some preferred embodiments, a substantially purepolypeptide typically comprises about 50%, 60%, 70%, 80%, 90% 95%, 96%,97%, 98%, 99% or 99.5% weight/weight of a protein sample. Polypeptidepurity or homogeneity is indicated by a number of methods well known inthe art, such as agarose or polyacrylamide gel electrophoresis of asample, followed by visualizing a single polypeptide band upon stainingthe gel. For certain purposes, higher resolution can be achieved byusing HPLC or other methods well known in the art.

Further, as used herein, the term “purified” does not require absolutepurity; rather, it is intended as a relative definition. Purification ofstarting material or natural material to at least one order ofmagnitude, preferably two or three orders, and more preferably four orfive orders of magnitude is expressly contemplated. Alternatively,purification may be expressed as “at least” a percent purity relative toheterologous polynucleotides (DNA, RNA or both) or polypeptides. As apreferred embodiment, the polynucleotides or polypeptides of the presentinvention are at least; 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, 96%, 96%, 98%, 99%, 99.5% or 100% pure relative to heterologouspolynucleotides or polypeptides. As a further preferred embodiment thepolynucleotides or polypeptides have an “at least” purity ranging fromany number, to the thousandth position, between 90% and 100% (e.g., atleast 99.995% pure) relative to heterologous polynucleotides orpolypeptides. Additionally, purity of the polynucleotides orpolypeptides may be expressed as a percentage (as described above)relative to all materials and compounds other than the carrier solution.Each number, to the thousandth position, may be claimed as individualspecies of purity.

The term “isolated” requires that the material be removed from itsoriginal environment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or DNA or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotide could be part of a vector and/or such polynucleotide orpolypeptide could be part of a composition, and still be isolated inthat the vector or composition is not part of its natural environment.

Specifically excluded from the definition of “isolated” are: naturallyoccurring chromosomes (e.g., chromosome spreads), artificial chromosomelibraries, genomic libraries, and cDNA libraries that exist either as anin vitro nucleic acid preparation or as a transfected/transformed hostcell preparation, wherein the host cells are either an in vitroheterogeneous preparation or plated as a heterogeneous population ofsingle colonies. Also specifically excluded are the above librarieswherein a 5′ EST makes up less than 5% (or alternatively 1%, 2%, 3%, 4%,10%, 25%, 50%, 75%, or 90%, 95%, or 99%) of the number of nucleic acidinserts in the vector molecules. Further specifically excluded are wholecell genomic DNA or whole cell RNA preparations (including said wholecell preparations which are mechanically sheared or enzymaticallydigested). Further specifically excluded are the above whole cellpreparations as either an in vitro preparation or as a heterogeneousmixture separated by electrophoresis (including blot transfers of thesame) wherein the polynucleotide of the invention have not been furtherseparated from the heterologous polynucleotides in the electrophoresismedium (e.g., further separating by excising a single band from aheterogeneous band population in an agarose gel or nylon blot).

The term “primer” denotes a specific oligonucleotide sequence which iscomplementary to a target nucleotide sequence and used to hybridize tothe target nucleotide sequence. A primer serves as an initiation pointfor nucleotide polymerization catalyzed by DNA polymerase, RNApolymerase, or reverse transcriptase.

The term “probe” denotes a defined nucleic acid segment that can be usedto identify a specific polynucleotide sequence present in a sample, saidnucleic acid segment comprising a nucleotide sequence complementary tothe specific polynucleotide sequence to be identified.

The term “polypeptide” refers to a polymer of amino acids without regardto the length of the polymer. Thus, peptides, oligopeptides, andproteins are included within the definition of polypeptide. This termalso does not specify or exclude post-expression modifications ofpolypeptides. For example, polypeptides that include the covalentattachment of glycosyl groups, acetyl groups, phosphate groups, lipidgroups and the like are expressly encompassed by the term polypeptide.Also included within the definition are polypeptides which contain oneor more analogs of an amino acid (including, for example, non-naturallyoccurring amino acids, amino acids which only occur naturally in anunrelated biological system, modified amino acids from mammalian systemsetc.), polypeptides with substituted linkages, as well as othermodifications known in the art, both naturally occurring andnon-naturally occurring.

Without being limited by theory, the compounds/polypeptides of theinvention are capable of modulating the partitioning of dietary lipidsbetween the liver and peripheral tissues, and thus of treating “diseasesinvolving the partitioning of dietary lipids between the liver andperipheral tissues.” The term “peripheral tissues” is meant to includemuscle and adipose tissue. In preferred embodiments, thecompounds/polypeptides of the invention partition the dietary lipidstoward the muscle. In alternative preferred embodiments, the dietarylipids are partitioned toward the adipose tissue. In other preferredembodiments, the dietary lipids are partitioned toward the liver. In yetother preferred embodiments, the compounds/polypeptides of the inventionincrease or decrease the oxidation of dietary lipids, preferably freefatty acids (FFA) by the muscle. Dietary lipids include, but are notlimited to triglycerides and free fatty acids.

Preferred diseases believed to involve the partitioning of dietarylipids include obesity and obesity-related diseases and disorders suchas obesity, impaired glucose tolerance, insulin resistance,atherosclerosis, atheromatous disease, heart disease, hypertension,stroke, Syndrome X, Non-Insulin Dependent Diabetes Mellitus (NIDDM, orType II diabetes) and Insulin Dependent Diabetes Mellitus (IDDM or TypeI diabetes). Diabetes-related complications to be treated by the methodsof the invention include microangiopathic lesions, ocular lesions,retinopathy, neuropathy, and renal lesions. Heart disease includes, butis not limited to, cardiac insufficiency, coronary insufficiency, andhigh blood pressure. Other obesity-related disorders to be treated bycompounds of the invention include hyperlipidemia and hyperuricemia. Yetother obesity-related diseases or disorders of the invention includecachexia, wasting, AIDS-related weight loss, cancer-related weight loss,anorexia, and bulimia.

The term “heterologous”, when used herein, is intended to designate anypolypeptide or polynucleotide other than a GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B polypeptide or a polynucleotide encoding a GMG-3,GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide of the presentinvention.

The terms “comprising”, “consisting of” and “consisting essentially of”are defined according to their standard meaning. A defined meaning setforth in the M.P.E.P. controls over a defined meaning in the art and adefined meaning set forth in controlling Federal Circuit case lawcontrols over a meaning set forth in the M.P.E.P. With this in mind, theterms may be substituted for one another throughout the instantapplication in order to attach the specific meaning associated with eachterm.

The term “host cell recombinant for” a particular polynucleotide of thepresent invention, means a host cell that has been altered by the handsof man to contain said polynucleotide in a way not naturally found insaid cell. For example, said host cell may be transiently or stablytransfected or transduced with said polynucleotide of the presentinvention.

The term “obesity” as used herein is defined in the WHO classificationsof weight (Kopelman (2000) Nature 404:635643). Underweight is less than18.5 (thin); Healthy is 18.5-24.9 (normal); grade 1 overweight is25.0-29.9 (overweight); grade 2 overweight is 30.0-39.0 (obesity); grade3 overweight is greater than or equal to 40.0 BMI. BMI is body massindex (morbid obesity) and is kg/m². Waist circumference can also beused to indicate a risk of metabolic complications where in men acircumference of greater than or equal to 94 cm indicates an increasedrisk, and greater than or equal to 102 cm indicates a substantiallyincreased risk. Similarly for women, greater than or equal to 88 cmindicates an increased risk, and greater than or equal to 88 cmindicates a substantially increased risk. The waist circumference ismeasured in cm at midpoint between lower border of ribs and upper borderof the pelvis. Other measures of obesity include, but are not limitedto, skinfold thickness which is a measurement in cm of skinfoldthickness using calipers, and bioimpedance, which is based on theprinciple that lean mass conducts current better than fat mass becauseit is primarily an electrolyte solution; measurement of resistance to aweak current (impedance) applied across extremities provides an estimateof body fat using an empirically derived equation.

The term “diabetes” as used herein is intended to encompass the usualdiagnosis of diabetes made from any of the methods included, but notlimited to, the following list: symptoms of diabetes (eg. polyuria,polydipsia, polyphagia) plus casual plasma glucose levels of greaterthan or equal to 200 mg/dl, wherein casual plasma glucose is defined anytime of the day regardless of the timing of meal or drink consumption; 8hour fasting plasma glucose levels of less than or equal to 126 mg/dl;and plasma glucose levels of greater than or equal to 200 mg/dl 2 hoursfollowing oral administration of 75 g anhydrous glucose dissolved inwater.

The term “impaired glucose tolerance (IGT)” as used herein is intendedto indicate that condition associated with insulin-resistance that isintermediate between frank, NIDDM and normal glucose tolerance (NGT). Ahigh percentage of the IGT population is known to progress to NIDDMrelative to persons with normal glucose tolerance (Sad et al., New EnglJ Med 1988; 319:1500-6). Thus, by providing therapeutics and methods forreducing or preventing IGT, i.e., for normalizing insulin resistance,the progression to NIDDM can be delayed or prevented. IGT is diagnosedby a procedure wherein an affected person's postprandial glucoseresponse is determined to be abnormal as assessed by 2-hour postprandialplasma glucose levels. In this test, a measured amount of glucose isgiven to the patient and blood glucose levels measured regularintervals, usually every half hour for the first two hours and everyhour thereafter. In a “normal” or non-IGT individual, glucose levelsrise during the first two hours to a level less than 140 mg/dl and thendrop rapidly. In an IGT individual, the blood glucose levels are higherand the drop-off level is at a slower rate.

The term “Insulin-Resistance Syndrome” as used herein is intended toencompass the cluster of abnormalities resulting from an attempt tocompensate for insulin resistance that sets in motion a series of eventsthat play an important role in the development of both hypertension andcoronary artery disease (CAD), such as premature atheroscleroticvascular disease. Increased plasma triglyceride and decreasedHDL-cholesterol concentrations, conditions that are known to beassociated with CAD, have also been reported to be associated withinsulin resistance. Thus, by providing therapeutics and methods forreducing or preventing insulin resistance, the invention providesmethods for reducing and/or preventing the appearance ofinsulin-resistance syndrome.

The term “polycystic ovary syndrome (PCOS)” as used herein is intendedto designate that etiologically unassigned disorder of premenopausalwomen, affecting 5-10% of this population, characterized byhyperandrogenism, chronic anovulation, defects in insulin action,insulin secretion, ovarian steroidogenesis and fibrinolysis. Women withPCOS frequently are insulin resistant and at increased risk to developglucose intolerance or NIDDM in the third and fourth decades of life(Dunaif et al. (1996) J Clin Endocrinol Metab 81:3299). Hyperandrogenismalso is a feature of a variety of diverse insulin-resistant states, fromthe type A syndrome, through leprechaunism and lipoatrophic diabetes, tothe type B syndrome, when these conditions occur in premenopausal women.It has been suggested that hyperinsulinemia per se causeshyperandrogenism. Insulin-sensitizing agents, e.g., troglitazone, havebeen shown to be effective in PCOS and that, in particular, the defectsin insulin action, insulin secretion, ovarian steroidogenosis andfibrinolysis are improved (Ehrman et al. (1997) J Clin Invest 100:1230),such as in insulin-resistant humans.

The term “insulin resistance” as used herein is intended to encompassthe usual diagnosis of insulin resistance made by any of a number ofmethods, including but not restricted to: the intravenous glucosetolerance test or measurement of the fasting insulin level. It is wellknown that there is an excellent correlation between the height of thefasting insulin level and the degree of insulin resistance. Therefore,one could use elevated fasting insulin levels as a surrogate marker forinsulin resistance for the purpose of identifying which normal glucosetolerance (NGT) individuals have insulin resistance. Another way to dothis is to follow the approach as disclosed in The New England Journalof Medicine, No. 3, pp. 1188 (1995), i.e. to select obesity as aninitial criterion for entry into the treatment group. Some obesesubjects have impaired glucose tolerance (IGT) while others have normalglucose tolerance (NGT). Since essentially all obese subjects areinsulin resistant, i.e. even the NGT obese subjects are insulinresistant and have fasting hyperinsulinemia. Therefore, the target ofthe treatment according to the present invention can be defined as NGTindividuals who are obese or who have fasting hyperinsulinemia, or whohave both.

A diagnosis of insulin resistance can also be made using the euglycemicglucose clamp test. This test involves the simultaneous administrationof a constant insulin infusion and a variable rate glucose infusion.During the test, which lasts 3-4 hours, the plasma glucose concentrationis kept constant at euglycemic levels by measuring the glucose levelevery 5-10 minutes and then adjusting the variable rate glucose infusionto keep the plasma glucose level unchanged. Under these circumstances,the rate of glucose entry into the bloodstream is equal to the overallrate of glucose disposal in the body. The difference between the rate ofglucose disposal in the basal state (no insulin infusion) and theinsulin infused state, represents insulin mediated glucose uptake. Innormal individuals, insulin causes brisk and large increase in overallbody glucose disposal, whereas in NIDDM subjects, this effect of insulinis greatly blunted, and is only 20-30% of normal. In insulin resistantsubjects with either IGT or NGT, the rate of insulin stimulated glucosedisposal is about half way between normal and NIDDM. For example, at asteady state plasma insulin concentration of about 100 uU/ml (aphysiologic level) the glucose disposal rate in normal subjects is about7 mg/kg/min. In NIDDM subjects, it is about 2.5 mg/kg/min., and inpatients with IGT (or insulin resistant subjects with NGT) it is about4-5 mg/kg/min. This is a highly reproducible and precise test, and candistinguish patients within these categories. It is also known that assubjects become more insulin resistant, the fasting insulin level rises.There is an excellent positive correlation between the height of thefasting insulin level and the magnitude of the insulin resistance asmeasured by euglycemic glucose clamp tests and, therefore, this providesthe rationale for using fasting insulin levels as a surrogate measure ofinsulin resistance.

The term “agent acting on the partitioning of dietary lipids between theliver and peripheral tissues” refers to a compound or polypeptide of theinvention that modulates the partitioning of dietary lipids between theliver and the peripheral tissues as previously described. Preferably,the agent increases or decreases the oxidation of dietary lipids,preferably free fatty acids (FFA) by the muscle. Preferably the agentdecreases or increases the body weight of individuals or is used totreat or prevent an obesity-related disease or disorder such as obesity,impaired glucose tolerance, insulin resistance, atherosclerosis,atheromatous disease, heart disease, hypertension, stroke, Syndrome X,Non-Insulin Dependent Diabetes Mellitus (NIDDM, or Type II diabetes) andInsulin Dependent Diabetes Mellitus (IDDM or Type I diabetes).Diabetes-related complications to be treated by the methods of theinvention include microangiopathic lesions, ocular lesions, retinopathy,neuropathy, renal lesions. Heart disease includes, but is not limitedto, cardiac insufficiency, coronary insufficiency, and high bloodpressure. Other obesity-related disorders to be treated by compounds ofthe invention include hyperlipidemia and hyperuricemia. Yet otherobesity-related diseases or disorders of the invention include cachexia,wasting, AIDS-related weight loss, cancer-related weight loss, anorexia,and bulimia.

The terms “response to an agent acting on the partitioning of dietarylipids between the liver and peripheral tissues” refer to drug efficacy,including but not limited to, ability to metabolize a compound, abilityto convert a pro-drug to an active drug, and the pharmacokinetics(absorption, distribution, elimination) and the pharmacodynamics(receptor-related) of a drug in an individual.

The terms “side effects to an agent acting on the partitioning ofdietary lipids between the liver and peripheral tissues” refer toadverse effects of therapy resulting from extensions of the principalpharmacological action of the drug or to idiosyncratic adverse reactionsresulting from an interaction of the drug with unique host factors.“Side effects to an agent acting on the partitioning of dietary lipidsbetween the liver and peripheral tissues” can include, but are notlimited to, adverse reactions such as dermatologic, hematologic orhepatologic toxicities and further includes gastric and intestinalulceration, disturbance in platelet function, renal injury, nephritis,vasomotor rhinitis with profuse watery secretions, angioneurotic edema,generalized urticaria, and bronchial asthma to laryngeal edema andbronchoconstriction, hypotension, and shock.

The term “GMG-3-, GMG-4-, Cluster 1-, GMG-6A-, or GMG-6B-relateddiseases and disorders” as used herein refers to any disease or disordercomprising an aberrant functioning of GMG-3, GMG-4, Cluster 1, GMG-6A,or GMG-6B, or which could be treated or prevented by modulating GMG-3,GMG-1, Cluster 1, GMG-6A, or GMG-6B levels or activity. “Aberrantfunctioning of GMG-3, GMG-1, Cluster 1, GMG-6A, or GMG-6B” includes, butis not limited to, aberrant levels of expression of GMG-3, GMG-4,Cluster 1, GMG-6A, or GMG-6B (either increased or decreased, butpreferably decreased), aberrant activity of GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B (either increased or decreased), and aberrantinteractions with ligands or binding partners (either increased ordecreased). By “aberrant” is meant a change from the type, or level ofactivity seen in normal cells, tissues, or patients, or seen previouslyin the cell, tissue, or patient prior to the onset of the illness. Inpreferred embodiments, these GMG-3-, GMG-4-, Cluster 1-, GMG-6A-, orGMG-6B-related diseases and disorders include obesity and themetabolic-related diseases and disorders described previously.

The term “cosmetic treatments” is meant to include treatments withcompounds or polypeptides of the invention that increase or decrease thebody mass of an individual where the individual is not clinically obeseor clinically thin. Thus, these individuals have a body mass index (BMI)below the cut-off for clinical obesity (e.g. below 25 kg/m²) and abovethe cut-off for clinical thinness (e.g. above 18.5 kg/m²). In addition,these individuals are preferably healthy (e.g. do not have anmetabolic-related disease or disorder of the invention). “Cosmetictreatments” are also meant to encompass, in some circumstances, morelocalized increases in adipose tissue, for example, gains or lossesspecifically around the waist or hips, or around the hips and thighs,for example. These localized gains or losses of adipose tissue can beidentified by increases or decreases in waist or hip size, for example.

The term “preventing” as used herein refers to administering a compoundprior to the onset of clinical symptoms of a disease or condition so asto prevent a physical manifestation of aberrations associated withobesity or GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B.

The term “treating” as used herein refers to administering a compoundafter the onset of clinical symptoms.

The term “in need of treatment” as used herein refers to a judgment madeby a caregiver (e.g. physician, nurse, nurse practitioner, etc in thecase of humans; veterinarian in the case of animals, including non-humanmammals) that an individual or animal requires or will benefit fromtreatment. This judgment is made based on a variety of factors that arein the realm of a caregiver's expertise, but that include the knowledgethat the individual or animal is ill, or will be ill, as the result of acondition that is treatable by the compounds of the invention.

The term “perceives a need for treatment” refers to a sub-clinicaldetermination that an individual desires to reduce weight for cosmeticreasons as discussed under “cosmetic treatment” above. The term“perceives a need for treatment” in other embodiments can refer to thedecision that an owner of an animal makes for cosmetic treatment of theanimal.

The term “individual” or “patient” as used herein refers to any animal,including mammals, preferably mice, rats, other rodents, rabbits, dogs,cats, swine, cattle, sheep, horses, or primates, and most preferablyhumans. The term may specify male or female or both, or exclude male orfemale.

The term “non-human animal” refers to any non-human vertebrate,including birds and more usually mammals, preferably primates, animalssuch as swine, goats, sheep, donkeys, horses, cats, dogs, rabbits orrodents, more preferably rats or mice. Both the terms “animal” and“mammal” expressly embrace human subjects unless preceded with the term“non-human”.

GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides are able tosignificantly reduce the postprandial response of plasma free fattyacids, glucose, and triglycerides in mammals fed a high fat/sucrosemeal, while not affecting levels of leptin, insulin or glucagon. Inaddition, GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptidesmodulate muscle free fatty acid oxidation in vitro and ex vivo,preferably increase oxidation. Further, GMG-3, GMG-4, Cluster 1, GMG-6A,or GMG-6B polypeptides of the invention modulate weight gain in mammalsthat are fed a high fat/sucrose diet.

The instant invention encompasses the use of GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B polypeptides in the partitioning of free fatty acid(FFA) and as an important new tool to control energy homeostasis. Of thetissues that can significantly remove lipids from circulation and causeFFA oxidation, muscle is believed to be quantitatively the mostimportant.

PREFERRED EMBODIMENTS OF THE INVENTION

I. GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B Polypeptides of theInvention

GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides have beenidentified that have measurable activity in vitro and in vivo. Theseactivities include, but are not limited to, modulation, preferablyreduction, of the postprandial response of plasma free fatty acids,glucose, and triglycerides in mammals fed a high fat/sucrose meal(Example 6), change, preferably an increase, in muscle free fatty acidoxidation in vitro and ex vivo (Example 10), and sustained weight lossin mammals on a high fat/sucrose diet. Other assays for GMG-3, GMG-4,Cluster 1, GMG-6A, or GMG-6B polypeptide activity in vitro and in vivoare also provided (Examples 2, 5, 7, 9, 11, for example), and equivalentassays can be designed by those with ordinary skill in the art.

The term “GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides”includes both the “full-length” polypeptide and fragments of the“full-length” GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides(although each of the above species may be particularly specified).

By “intact” or “full-length” GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bpolypeptides as used herein is meant the full-length polypeptidesequence of any GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide,from the N-terminal methionine to the C-terminal stop codon. Examples ofintact or full-length GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bpolypeptides are found in the sequence listing.

The term “metabolic-related activity” as used herein refers to at leastone, and preferably all, of the activities described herein for GMG-3,GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides. Assays for thedetermination of these activities are provided herein (e.g. Examples 2,5-7, 9-11), and equivalent assays can be designed by those with ordinaryskill in the art. Optionally, “metabolic-related activity” can beselected from the group consisting of lipid partitioning, lipidmetabolism, and insulin-like activity, or an activity within one ofthese categories. By “lipid partitioning” activity is meant the abilityto effect the location of dietary lipids among the major tissue groupsincluding, adipose tissue, liver, and muscle. GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B polypeptides of the invention play a role in thepartitioning of lipids to the muscle, liver or adipose tissue. By “lipidmetabolism” activity is meant the ability to influence the metabolism oflipids. GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides of theinvention have the ability to affect the level of free fatty acids inthe plasma as well as to modulate, preferably increase, the metabolismof lipids in the muscle through free fatty acid oxidation experiments(Examples 2, 6, 8, 9, 10) and to transiently affect the levels oftriglycerides in the plasma and the muscle (Examples 6, 8, 11). By“insulin-like” activity is meant the ability of GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B polypeptides to modulate the levels of glucose in theplasma. GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides do notsignificantly impact insulin levels but do impact glucose levelssimilarly to the effects of insulin (examples 7 & 8). These effects mayvary in the presence of the intact (full-length) GMG-3, GMG-4, Cluster1, GMG-6A, or GMG-6B polypeptides or are significantly greater in thepresence of the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptidefragments compared with the full-length GMG-3, GMG-4, Cluster 1, GMG-6A,or GMG-6B polypeptides.

The term “significantly greater” as used herein refers to a comparisonof the activity of a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bpolypeptide in an metabolic-related assay compared with untreated cellsin the same assay. By “significantly” as used herein is meantstatistically significant as it is typically determined by those withordinary skill in the art. For example, data are typically calculated asa mean±SEM, and a p-value≦0.05 is considered statistically significant.Statistical analysis is typically done using either the unpairedStudent's t test or the paired Student's t test, as appropriate in eachstudy. Examples of a significant change in activity as a result of thepresence of a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide ofthe invention compared to untreated cells include an increase or adecrease in a given parameter of at least 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%. One or more, but notnecessarily all, of the measurable parameters will change significantlyin the presence of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bpolypeptide as compared to untreated cells.

Representative “metabolic-related assays” are provided in the Examples.These assays include, but are not limited to, methods of measuring thepostprandial response, methods of measuring free fatty acid oxidation,and methods of measuring weight modulation. In preferred embodiments,the post-prandial response is measured in non-human animals, preferablymice. In preferred embodiments changes in dietary lipids are measured,preferably free fatty acids and/or triglycerides. In other embodiments,other physiologic parameters are measured including, but not limited to,levels of glucose, insulin, and leptin. In other preferred embodiments,free fatty acid oxidation is measured in cells in vitro or ex vivo,preferably in muscle cells or tissue of non-human animals, preferablymice. In yet other preferred embodiments weight modulation is measuredin human or non-human animals, preferably rodents (rats or mice),primates, canines, felines or procines, on a high fat/sucrose diet.Optionally, “metabolic-related activity” includes other activities notspecifically identified herein. In general, “measurable parameters”relating to obesity and the field of metabolic research can be selectedfrom the group consisting of free fatty acid levels, free fatty acidoxidation, triglyceride levels, glucose levels, insulin levels, leptinlevels, food intake, weight, leptin and lipoprotein binding, uptake anddegradation and lipolysis stimulated receptor (LSR) expression.

In these metabolic-related assays, preferred GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B polypeptides would cause a significant change in atleast one of the measurable parameters selected from the groupconsisting of post-prandial lipemia, free fatty acid levels,triglyceride levels, glucose levels, free fatty acid oxidation, andweight. Alternatively, preferred GMG-3, GMG-4, Cluster 1, GMG-6A, orGMG-6B polypeptides would have a significant change in at least one ofthe measurable parameters selected from the group consisting of anincrease in LSR activity, an increase in leptin activity and an increasein lipoprotein activity. By “LSR” activity is meant expression of LSR onthe surface of the cell, or in a particular conformation, as well as itsability to bind, uptake, and degrade leptin and lipoprotein. By “leptin”activity is meant its binding, uptake and degradation by LSR, as well asits transport across a blood brain barrier, and potentially theseoccurrences where LSR is not necessarily the mediating factor or theonly mediating factor. Similarly, by “lipoprotein” activity is meant itsbinding, uptake and degradation by LSR, as well as these occurrenceswhere LSR is not necessarily the mediating factor or the only mediatingfactor.

The invention is drawn, inter alia, to isolated, purified or recombinantGMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides. GMG-3, GMG-4,Cluster 1, GMG-6A, or GMG-6B polypeptides of the invention are usefulfor reducing or, using antagonists of GMG-3, GMG-4, Cluster 1, GMG-6A,or GMG-6B polypeptides, increasing body weight either as a cosmetictreatment or for treatment or prevention of metabolic-related diseasesand disorders. GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptidesare also useful inter alia in screening assays for agonists orantagonists of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptideactivity; for raising GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bpolypeptide-specific antibodies; and in diagnostic assays. When used forcosmetic treatments, or for the treatment or prevention ofmetabolic-related diseases, disorders or conditions, one or more GMG-3,GMG-4, Cluster 1, GMG-6A, or GMG-6B or GMG-3, GMG-4, Cluster 1, GMG-6A,or GMG-6B polypeptide fragments can be provided to a subject. Thus,various fragments of the full-length protein can be combined into a“cocktail” for use in the various treatment regimens.

The full-length GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG B polypeptide iscomprised of about four distinct regions including:

1. an N-terminal putative signal peptide sequence about from amino acids1-19 of SEQ ID NO: 2, 4, 8, or 10;

2. a unique region about from amino acids 20-25 of SEQ ID NO: 2, 4, 8,or 10;

3. a collagen-like region about from amino acids 26-196 of SEQ ID NO: 2or 4, from about amino acids 1-87 of SEQ ID NO: 6, from about aminoacids 26-193 of SEQ ID NO: 8, or from about amino acids 26-186 of SEQ IDNO: 10; and

4. a C-terminal globular C1q homology domain about from amino acids200-333 of SEQ ID NO: 2 or 4, about from amino acids 88-225 of SEQ IDNO: 6, about from amino acids 197-330 of SEQ ID NO: 8, or about fromamino acids 190-323 of SEQ ID NO: 10.

GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides of the inventioninclude variants, fragments, analogs and derivatives of the GMG-3,GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides described above,including modified GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bpolypeptides.

The GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides of thepresent invention are preferably provided in an isolated form, and maybe partially or substantially purified. A recombinantly produced versionof any one of the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bpolypeptides can be substantially purified by the one-step methoddescribed by Smith et al. ((1988) Gene 67:31-40) or by the methodsdescribed herein or known in the art Polypeptides of the invention alsocan be purified from natural or recombinant sources using antibodiesdirected against the polypeptides of the invention by methods known inthe art of protein purification.

Preparations of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptidesof the invention involving a partial purification of or selection forthe GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides are alsospecifically contemplated. These crude preparations are envisioned to bethe result of the concentration of cells expressing GMG-3, GMG-4,Cluster 1, GMG-6A, or GMG-6B polypeptides with perhaps a few additionalpurification steps, but prior to complete purification of the fragment.The cells expressing GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bpolypeptides are present in a pellet, they are lysed, or the crudepolypeptide is lyophilized, for example.

GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide fragments can beany integer in length from at least 6 consecutive amino acids to oneamino acid less than a full-length GMG-3, GMG-4, Cluster 1, GMG-6A, orGMG-6B polypeptide. Thus, for the polypeptides of SEQ ID NO: 2 or 4, aGMG-3 or GMG-4 polypeptide fragment can be any integer of consecutiveamino acids from 6 to 332, for example. The term “integer” is usedherein in its mathematical sense and thus representative integersinclude, but are not limited to: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174,175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188,189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202,203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230,231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244,245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258,259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272,273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286,287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300,301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314,315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328,329, 330, 331 and 332.

Each GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide fragment asdescribed above can be further specified in terms of its N-terminal andC-terminal positions. For example, every combination of a N-terminal andC-terminal position that fragments of from 6 contiguous amino acids toone amino acid less than the full-length polypeptide of SEQ ID NO: 2 or4 could occupy, on any given intact and contiguous full-lengthpolypeptide sequence of SEQ ID NO: 2 or 4 are included in the presentinvention. Thus, a 6 consecutive amino acid fragment could occupypositions selected from the group consisting of 1-6, 2-7, 3-8, 4-9,5-10, 6-11, 7-12, 8-13, 9-14, 10-15, 11-16, 12-17, 13-18, 14-19, 15-20,16-21, 17-22, 18-23, 19-24, 20-25, 21-26, 22-27, 23-28, 24-29, 25-30,26-31, 27-32, 28-33, 29-34, 30-35, 31-36, 32-37, 33-38, 34-39, 35-40,36-41, 3742, 38-43, 39-44, 40-45, 41-46, 4247, 43-48, 44-49, 45-50,46-51, 47-52, 48-53, 49-54, 50-55, 51-56, 52-57, 53-58, 54-59, 55-60,56-61, 57-62, 58-63, 59-64, 60-65, 61-66, 62-67, 63-68, 64-69, 65-70,66-71, 67-72, 68-73, 69-74, 70-75, 71-76, 72-77, 73-78, 74-79, 75-80,76-81, 77-82, 78-83, 79-84, 80-85, 81-86, 82-87, 83-88, 84-89, 85-90,86-91, 87-92, 88-93, 89-94, 90-95, 91-96, 92-97, 93-98, 94-99, 95-100,96-101, 97-102, 98-103, 99-104, 100-105, 101-106, 102-107, 103-108,104-109, 105-110, 106-111, 107-112, 108-113, 109-114, 110-115, 111-116,112-117, 113-118, 114-119, 115-120, 116-121, 117-122, 118-123, 119-124,120-125, 121-126, 122-127, 123-128, 124-129, 125-130, 126-131, 127-132,128-133, 129-134, 130-135, 131-136, 132-137, 133-138, 134-139, 135-140,136-141, 137-142, 138-143, 139-144, 140-145, 141-146, 142-147, 143-148,144-149, 145-150, 146-151, 147-152, 148-153, 149-154, 150-155, 151-156,152-157, 153-158, 154-159, 155-160, 156-161, 157-162, 158-163, 159-164,160-165, 161-166, 162-167, 163-168, 164-169, 165-170, 166-171, 167-172,168-173, 169-174, 170-175, 171-176, 172-177, 173-178, 174-179, 175-180,176-181, 177-182, 178-183, 179-184, 180-185, 181-186, 182-187, 183-188,184-189, 185-190, 186-191, 187-192, 188-193, 189-194, 190-195, 191-196,192-197, 193-198, 194-199, 195-200, 196-201, 197-202, 198-203, 199-204,200-205, 201-206, 202-207, 203-208, 204-209, 205-210, 206-211, 207-212,208-213, 209-214, 210-215, 211-216, 212-217, 213-218, 214-219,215-220,216-221, 217-222, 218-223, 219-224, 220-225, 221-226, 222-227,223-228, 224-229, 225-230, 226-231, 227-232, 228-233, 229-234, 230-235,231-236, 232-237, 233-238, 234-239, 235-240, 236-241, 237-242, 238-243,240-245, 241-246, 242-247, 243-248, 244-249, 245-250, 246-251, 247-252,248-253, 249-254, 250-255, 251-256, 252-257, 253-258, 254-259, 255-260,256-261, 257-262, 258-263, 259-264, 260-265, 261-266, 262-267, 263-268,264-269, 265-270, 266-271, 267-272, 268-273, 269-274, 270-275, 271-276,272-277, 273-278, 274-279, 275-280, 276-281, 277-282, 278-283, 279-284,280-285, 281-286, 282-287, 283-288, 284-289, 285-290, 286-291, 287-292,288-293, 289-294, 290-295, 291-296, 292-297, 293-298, 294-299, 295-300,296-301, 297-302, 298-303, 299-304, 300-305, 301-306, 302-307, 303-308,304-309, 305-310, 306-311, 307-312, 308-313, 309-314, 310-315, 311-316,312-317, 313-318, 314-319, 315-320, 316-321, 317-322, 318-323, 319-324,320-325, 321-326, 322-327, 323-328, 324-329, 325-330, 326-331, 327-332and 328-333 of a 333 consecutive amino acid fragment. A 327 consecutiveamino acid fragment could occupy positions selected from the groupconsisting of 1-327, 2-328, 3-329, 4-330, 5-331, 6-332 and 7-333.Similarly, the positions occupied by all the other fragments of sizesbetween 6 amino acids and 332 amino acids in SEQ ID NO: 2 or 4, by allthe other fragments of sizes between 6 amino acids and 224 amino acidsin SEQ ID NO: 6, by all the other fragments of sizes between 6 aminoacids and 329 amino acids in SEQ ID NO: 8, and by all the otherfragments of sizes between 6 amino acids and 322 amino acids in SEQ IDNO: 10 are included in the present invention and can also be immediatelyenvisaged based on these two examples and therefore, are notindividually listed solely for the purpose of not unnecessarilylengthening the specification. Furthermore, the positions occupied byfragments of 6 to 332 consecutive amino acids in SEQ ID NO: 2 or 4, byfragments of 6 to 224 consecutive amino acids in SEQ ID NO: 6, byfragments of 6 to 329 amino acids in SEQ ID NO: 8, and by fragments of 6to 322 amino acids in SEQ ID NO: 10 are included in the presentinvention and can also be immediately envisaged based on these twoexamples and therefore are not individually listed solely for thepurpose of not unnecessarily lengthening the specification. In addition,the positions occupied by fragments of 6 consecutive amino acids to 1amino acid less than any other full-length GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B polypeptide can also be envisaged based on these twoexamples and therefore are not individually listed solely for thepurpose of not unnecessarily lengthening the specification.

The GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides of thepresent invention may alternatively be described by the formula “n to c”(inclusive); where “n” equals the N-terminal most amino acid position(as defined by the sequence listing) and “c” equals the C-terminal mostamino acid position (as defined by the sequence listing) of thepolypeptide; and further where “n” equals an integer between 1 and thenumber of amino acids of the full-length polypeptide sequence of thepresent invention minus 6; and where “c” equals an integer between 7 andthe number of amino acids of the full-length polypeptide sequence; andwhere “n” is an integer smaller then “c” by at least 6. Therefore, forthe sequences provided in SEQ ID NO: 2 or 4, “n” is any integer selectedfrom the list consisting of: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102,103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116,117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130,131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144,145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158,159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172,173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186,187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200,201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214,215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228,229, 230, 231, 232, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243,244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257,258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271,272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285,286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299,300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313,314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326 or 327and “c” is any integer selected from the group consisting of: 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169,170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183,184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197,198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211,212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,226, 227, 228, 229, 230, 231, 232, 234, 235, 236, 237, 238, 239, 240,241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254,255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268,269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282,283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296,297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310,311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324,325, 326, 327, 328, 329, 330, 331, 332 or 333. Every combination of “n”and “c” positions are included as specific embodiments of the invention.Moreover, the formula “n” to “c” may be modified as “n1-n2” to “c1-c2”,wherein “n1-n2” and “c1-c2” represent positional ranges selected fromany two integers above which represent amino acid positions of thesequence listing. Alternative formulas include “n1-n2” to “c” and “n” to“c1-c2”. In a preferred embodiment, GMG-3 or GMG-4 polypeptide fragmentsof the invention may be described by the formula where n1=20, n2=202,and c=333 of SEQ ID NO: 2 or 4; Cluster 1 polypeptide fragments of theinvention may be described by the formula n1=1, n2=94, and c=225 of SEQID NO: 6; GMG-6A polypeptide fragments of the invention may be describedby the formula n1=20, n2=199, and c=330 of SEQ ID NO: 8; or GMG-6Bpolypeptide fragments of the invention may be described by the formulawhere n1=20, n2=192, and c=323 of SEQ ID NO: 10.

Furthermore, the positions occupied by polypeptides of 6 to 225consecutive amino acids on SEQ ID NO: 6, by polypeptides of 6 to 330consecutive amino acids on SEQ ID NO:8, or by polypeptides of 6 to 323consecutive amino acids on SEQ ID NO: 10, are included in the presentinvention and can also be immediately envisaged based on these twoexamples and therefore are not individually listed solely for thepurpose of not unnecessarily lengthening the specification. In addition,the positions occupied by fragments of 6 consecutive amino acids to 1amino acid less than full-length Cluster 1, GMG-6A, or GMG-6Bpolypeptide can also be envisaged based on these two examples andtherefore are not individually listed solely for the purpose of notunnecessarily lengthening the specification.

In preferred embodiments, GMG-3 or GMG-4 polypeptide fragments havingactivity are selected from amino acids 20-333, 43-333, 44-333, 45-333,46-333, 47-333, 48-333, 49-333, 50-333, 51-333, 52-333, 53-333, 54-333,55-333, 56-333, 57-333, 58-333, 59-333, 60-333, 61-333, 62-333, 63-333,64-333, 65-333, 66-333, 67-333, 68-333, 69-333, 70-333, 71-333, 72-333,73-333, 74-333, 75-333, 76-333, 77-333, 78-333, 79-333, 80-333, 81-333,82-333, 83-333, 84-333, 85-333, 86-333, 87-333, 88-333, 89-333, 90-333,91-333, 92-333, 93-333, 94-333, 95-333, 96-333, 97-333, 98-333, 99-333,100-333, 101-333, 102-333, 103-333, 104-333, 105-333, 106-333, 107-333,108-333, 109-333, 110-333, 111-333, 112-333, 113-333, 114-333, 115-333,116-333, 117-333, 118-333, 119-333, 120-333, 121-333, 122-333, 123-333,124-333, 125-333, 126-333, 127-333, 128-333, 129-333, 130-333, 131-333,132-333, 133-333, 134-333, 135-333, 136-333, 137-333, 138-333, 139-333,140-333, 141-333, 142-333, 143-333, 144-333, 145-333, 146-333, 147-333,148-333, 149-333, 150-333, 151-333, 152-333, 153-333, 154-333, 155-333,156-333, 157-333, 158-333, 159-333, 160-333, 161-333, 162-333, 163-333,164-333, 165-333, 166-333, 167-333, 168-333, 169-333, 170-333, 171-333,172-333, 173-333, 174-333, 175-333, 176-333, 177-333, 178-333, 179-333,180-333, 181-333, 182-333, 183-333, 184-333, 185-333, 186-333, 187-333,188-333, 189-333, 190-333, 191-333, 192-333, 193-333, 194-333, 195-333,196-333, 197-333, 198-333, 199-333, 200-333, 201-333 or 202-333 of SEQID NO: 2 or 4. In other preferred embodiments, Cluster 1 polypeptidefragments having activity are selected from amino acids 1-225, 2-225,3-225, 4-225, 5-225, 6-225, 7-225, 8-225, 9-225, 10-225, 11-225, 12-225,13-225, 14-225, 15-225, 16-225, 17-225, 18-225, 19-225, 20-225, 21-225,22-225, 23-225, 24-225, 25-225, 26-225, 27-225, 28-225, 29-225, 30-225,31-225, 32-225, 33-225, 34-225, 35-225, 36-225, 37-225, 38-225, 39-225,40-225, 41-225, 42-225, 43-225, 44-225, 45-225, 46-225, 47-225, 48-225,49-225, 50-225, 51-225, 52-225, 53-225, 54-225, 55-225, 56-225, 57-225,58-225, 59-225, 60-225, 61-225, 62-225, 63-225, 64-225, 65-225, 66-225,67-225, 68-225, 69-225, 70-225, 71-225, 72-225, 73-225, 74-225, 75-225,76-225, 77-225, 78-225, 79-225, 80-225, 81-225, 82-225, 83-225, 84-225,85-225, 86-225, 87-225, 88-225, 89-225, 90-225, 91-225, 92-225, 93-225or 94-225 of SEQ ID NO: 6. In other preferred embodiments, GMG-6Apolypeptide fragments having activity are selected from amino acids20-330, 43-330, 44-330, 45-330, 46-330, 47-330, 48-330, 49-330, 50-330,51-330, 52-330, 53-330, 54-330, 55-330, 56-330, 57-330, 58-330, 59-330,60-330, 61-330, 62-330, 63-330, 64-330, 65-330, 66-330, 67-330, 68-330,69-330, 70-330, 71-330, 72-330, 73-330, 74-330, 75-330, 76-330, 77-330,78-330, 79-330, 80-330, 81-330, 82-330, 83-330, 84-330, 85-330, 86-330,87-330, 88-330, 89-330, 90-330, 91-330, 92-330, 93-330, 94-330, 95-330,96-330, 97-330, 98-330, 99-330, 100-330, 101-330, 102-330, 103-330,104-330, 105-330, 106-330, 107-330, 108-330, 109-330, 110-330, 111-330,112-330, 113-330, 114-330, 115-330, 116-330, 117-330, 118-330, 119-330,120-330, 121-330, 122-330, 123-330, 124-330, 125-330, 126-330, 127-330,128-330, 129-330, 130-330, 131-330, 132-330, 133-330, 134-330, 135-330,136-330, 137-330, 138-330, 139-330, 140-330, 141-330, 142-330, 143-330,144-330, 145-330, 146-330, 147-330, 148-330, 149-330, 150-330, 151-330,152-330, 153-330, 154-330, 155-330, 156-330, 157-330, 158-330, 159-330,160-330, 161-330, 162-330, 163-330, 164-330, 165-330, 166-330, 167-330,168-330, 169-330, 170-330, 171-330, 172-330, 173-330, 174-330, 175-330,176-330, 177-330, 178-330, 179-330, 180-330, 181-330, 182-330, 183-330,184-330, 185-330, 186-330, 187-330, 188-330, 189-330, 190-330, 191-330,192-330, 193-330, 194-330, 195-330, 196-330, 197-330, 198-330 or 199-330of SEQ ID NO: 8. In other preferred embodiments, GMG-6B polypeptidefragments having activity are selected from amino acids 20-323, 43-323,44-323, 45-323, 46-323, 47-323, 48-323, 49-323, 50-323, 51-323, 52-323,53-323, 54-323, 55-323, 56-323, 57-323, 58-323, 59-323, 60-323, 61-323,62-323, 63-323, 64-323, 65-323, 66-323, 67-323, 68-323, 69-323, 70-323,71-323, 72-323, 73-323, 74-323, 75-323, 76-323, 77-323, 78-323, 79-323,80-323, 81-323, 82-323, 83-323, 84-323, 85-323, 86-323, 87-323, 88-323,89-323, 90-323, 91-323, 92-323, 93-323, 94-323, 95-323, 96-323, 97-323,98-323, 99-323, 100-323, 101-323, 102-323, 103-323, 104-323, 105-323,106-323, 107-323, 108-323, 109-323, 110-323, 111-323, 112-323, 113-323,114-323, 115-323, 116-323, 117-323, 118-323, 119-323, 120-323, 121-323,122-323, 123-323, 124-323, 125-323, 126-323, 127-323, 128-323, 129-323,130-323, 131-323, 132-323, 133-323, 134-323, 135-323, 136-323, 137-323,138-323, 139-323, 140-323, 141-323, 142-323, 143-323, 144-323, 145-323,146-323, 147-323, 148-323, 149-323, 150-323, 151-323, 152-323, 153-323,154-323, 155-323, 156-323, 157-323, 158-323, 159-323, 160-323, 161-323,162-323, 163-323, 164-323, 165-323, 166-323, 167-323, 168-323, 169-323,170-323, 171-323, 172-323, 173-323, 174-323, 175-323, 176-323, 177-323,178-323, 179-323, 180-323, 181-323, 182-323, 183-323, 184-323, 185-323,186-323, 187-323, 188-323, 189-323, 190-323, 191-323 or 192-323 of SEQID NO: 10.

These specific embodiments, and other polypeptide and polynucleotidefragment embodiments described herein may be modified as being “atleast”, “equal to”, “equal to or less than”, “less than”, “at least______ but not greater than ______” or “from ______ to ______” aspecified size or specified N-terminal and/or C-terminal positions. Itis noted that all ranges used to describe any embodiment of the presentinvention are inclusive unless specifically set forth otherwise.

The present invention also provides for the exclusion of any individualfragment specified by N-terminal and C-terminal positions or of anyfragment specified by size in amino acid residues as described above. Inaddition, any number of fragments specified by N-terminal and C-terminalpositions or by size in amino acid residues as described above may beexcluded as individual species. Further, any number of fragmentsspecified by N-terminal and C-terminal positions or by size in aminoacid residues as described above may make up a polypeptide fragment inany combination and may optionally include non-GMG-3, -GMG-4, -Cluster1, -GMG-6A, or -GMG-6B polypeptide sequences as well.

In preferred embodiments, GMG-3 or GMG-4 polypeptide fragments havingactivity are selected from amino acids 20-333, 43-333, 44-333, 45-333,46-333, 47-333, 48-333, 49-333, 50-333, 51-333, 52-333, 53-333, 54-333,55-333, 56-333, 57-333, 58-333, 59-333, 60-333, 61-333, 62-333, 63-333,64-333, 65-333, 66-333, 67-333, 68-333, 69-333, 70-333, 71-333, 72-333,73-333, 74-333, 75-333, 76-333, 77-333, 78-333, 79-333, 80-333, 81-333,82-333, 83-333, 84-333, 85-333, 86-333, 87-333, 88-333, 89-333, 90-333,91-333, 92-333, 93-333, 94-333, 95-333, 96-333, 97-333, 98-333, 99-333,100-333, 101-333, 102-333, 103-333, 104-333, 105-333, 106-333, 107-333,108-333, 109-333, 110-333, 111-333, 112-333, 113-333, 114-333, 115-333,116-333, 117-333, 118-333, 119-333, 120-333, 121-333, 122-333, 123-333,124-333, 125-333, 126-333, 127-333, 128-333, 129-333, 130-333, 131-333,132-333, 133-333, 134-333, 135-333, 136-333, 137-333, 138-333, 139-333,140-333, 141-333, 142-333, 143-333, 144-333, 145-333, 146-333, 147-333,148-333, 149-333, 150-333, 151-333, 152-333, 153-333, 154-333, 155-333,156-333, 157-333, 158-333, 159-333, 160-333, 161-333, 162-333, 163-333,164-333, 165-333, 166-333, 167-333, 168-333, 169-333, 170-333, 171-333,172-333, 173-333, 174-333, 175-333, 176-333, 177-333, 178-333, 179-333,180-333, 181-333, 182-333, 183-333, 184-333, 185-333, 186-333, 187-333,188-333, 189-333, 190-333, 191-333, 192-333, 193-333, 194-333, 195-333,196-333, 197-333, 198-333, 199-333, 200-333, 201-333 or 202-333 of SEQID NO: 2 or 4. In other preferred embodiments, Cluster 1 polypeptidefragments having activity are selected from amino acids 1-225, 2-225,3-225, 4-225, 5-225, 6-225, 7-225, 8-225, 9-225, 10-225, 11-225, 12-225,13-225, 14-225, 15-225, 16-225, 17-225, 18-225, 19-225, 20-225, 21-225,22-225, 23-225, 24-225, 25-225, 26-225, 27-225, 28-225, 29-225, 30-225,31-225, 32-225, 33-225, 34-225, 35-225, 36-225, 37-225, 38-225, 39-225,40-225, 41-225, 42-225, 43-225, 44-225, 45-225, 46-225, 47-225, 48-225,49-225, 50-225, 51-225, 52-225, 53-225, 54-225, 55-225, 56-225, 57-225,58-225, 59-225, 60-225, 61-225, 62-225, 63-225, 64-225, 65-225, 66-225,67-225, 68-225, 69-225, 70-225, 71-225, 72-225, 73-225, 74-225, 75-225,76-225, 77-225, 78-225, 79-225, 80-225, 81-225, 82-225, 83-225, 84-225,85-225, 86-225, 87-225, 88-225, 89-225, 90-225, 91-225, 92-225, 93-225or 94-225 of SEQ ID NO: 6. In other preferred embodiments, GMG-6Apolypeptide fragments having activity are selected from amino acids20-330, 43-330, 44-330, 45-330, 46-330, 47-330, 48-330, 49-330, 50-330,51-330, 52-330, 53-330, 54-330, 55-330, 56-330, 57-330, 58-330, 59-330,60-330, 61-330, 62-330, 63-330, 64-330, 65-330, 66-330, 67-330, 68-330,69-330, 70-330, 71-330, 72-330, 73-330, 74-330, 75-330, 76-330, 77-330,78-330, 79-330, 80-330, 81-330, 82-330, 83-330, 84-330, 85-330, 86-330,87-330, 88-330, 89-330, 90-330, 91-330, 92-330, 93-330, 94-330; 95-330,96-330, 97-330, 98-330, 99-330, 100-330, 101-330, 102-330, 103-330,104-330, 105-330, 106-330, 107-330, 108-330, 109-330, 110-330, 111-330,112-330, 113-330, 114-330, 115-330, 116-330, 117-330, 118-330, 119-330,120-330, 121-330, 122-330, 123-330, 124-330, 125-330, 126-330, 127-330,128-330, 129-330, 130-330, 131-330, 132-330, 133-330, 134-330, 135-330,136-330, 137-330, 138-330, 139-330, 140-330, 141-330, 142-330, 143-330,144-330, 145-330, 146-330, 147-330, 148-330, 149-330, 150-330, 151-330,152-330, 153-330, 154-330, 155-330, 156-330, 157-330, 158-330, 159-330,160-330, 161-330, 162-330, 163-330, 164-330, 165-330, 166-330, 167-330,168-330, 169-330, 170-330, 171-330, 172-330, 173-330, 174-330, 175-330,176-330, 177-330, 178-330, 179-330, 180-330, 181-330, 182-330, 183-330,184-330, 185-330, 186-330, 187-330, 188-330, 189-330, 190-330, 191-330,192-330, 193-330, 194-330, 195-330, 196-330, 197-330, 198-330 or 199-330of SEQ ID NO: 8. In other preferred embodiments, GMG-6B polypeptidefragments having activity are selected from amino acids 20-323, 43-323,44-323, 45-323, 46-323, 47-323, 48-323, 49-323, 50-323, 51-323, 52-323,53-323, 54-323, 55-323, 56-323, 57-323, 58-323, 59-323, 60-323, 61-323,62-323, 63-323, 64-323, 65-323, 66-323, 67-323, 68-323, 69-323, 70-323,71-323, 72-323, 73-323, 74-323, 75-323, 76-323, 77-323, 78-323, 79-323,80-323, 81-323, 82-323, 83-323, 84-323, 85-323, 86-323, 87-323, 88-323,89-323, 90-323, 91-323, 92-323, 93-323, 94-323, 95-323, 96-323, 97-323,98-323, 99-323, 100-323, 101-323, 102-323, 103-323, 104-323, 105-323,106-323, 107-323, 108-323, 109-323, 110-323, 111-323, 112-323, 113-323,114-323, 115-323, 116-323, 117-323, 118-323, 119-323, 120-323, 121-323,122-323, 123-323, 124-323, 125-323, 126-323, 127-323, 128-323, 129-323,130-323, 131-323, 132-323, 133-323, 134-323, 135-323, 136-323, 137-323,138-323, 139-323, 140-323, 141-323, 142-323, 143-323, 144-323, 145-323,146-323, 147-323, 148-323, 149-323, 150-323, 151-323, 152-323, 153-323,154-323, 155-323, 156-323, 157-323, 158-323, 159-323, 160-323, 161-323,162-323, 163-323, 164-323, 165-323, 166-323, 167-323, 168-323, 169-323,170-323, 171-323, 172-323, 173-323, 174-323, 175-323, 176-323, 177-323,178-323, 179-323, 180-323, 181-323, 182-323, 183-323, 184-323, 185-323,186-323, 187-323, 188-323, 189-323, 190-323, 191-323 or 192-323 of SEQID NO: 10.

In more preferred embodiments, said GMG-3 or GMG-4 polypeptide fragmentscomprised of all or part of the C-terminal globular C1q homology domainand having activity are selected from amino acids 20-333, 43-333,45-333, 46-333, 50-333, 53-333, 61-333, 67-333, 74-333, 75-333, 77-333,81-333, 82-333, 86-333, 89-333, 95-333, 100-333, 104-333, 109-333,113-333, 116-333, 125-333, 128-333, 140-333, 160-333, 164-333, 179-333,182-333, 185-333, 188-333, 191-333, 193-333, 201-333, 202-333, 227-333,252-333, 252-267,252-317, 256-267,256-317, or 304-317 of SEQ ID NO: 2 or4. In other more preferred embodiments, said Cluster 1 polypeptidefragments having activity are selected from amino acids 1-225, 5-225,8-225, 17-225, 20-225, 32-225, 52-225, 56-225, 71-225, 74-225, 77-225,80-225, 83-225, 85-225, 93-225, 94-225, 119-225, 144-225, 144-159,144-209, 148-159, 148-209, or 196-209 of SEQ ID NO: 6. In other morepreferred embodiments, said GMG-6A polypeptide fragments having activityare selected from amino acids 20-330, 43-330, 45-330, 46-330, 50-333,53-330, 64-330, 68-330, 71-330, 72-330, 75-330, 78-330, 79-330, 83-330,86-330, 92-330, 97-330, 101-330, 122-330, 125-330, 146-330, 157-330,161-330, 176-330, 179-330, 182-330, 185-330, 188-330, 190-330, 198-330,199-330,224-330, 249-330, 249-264, 249-314, 253-264,253-314 or 301-314of SEQ ID NO: 8. In other more preferred embodiments, said GMG-6Bpolypeptide fragments having activity are selected from amino acids20-323, 43-323, 46-323, 57-323, 61-323, 64-323, 65-323, 68-323, 71-323,72-323, 76-323, 79-323, 85-323, 90-323, 94-323, 115-323, 118-323,139-323, 150-323, 154-323, 169-323, 172-323, 175-323, 178-323, 181-323,183-323, 191-323, 192-323, 217-323, 242-323, 242-257, 242-307, 246-257,246-307, or 294-307 of SEQ ID NO: 10.

In yet more preferred embodiments, said GMG-3 or GMG-4 polypeptidefragments comprised of all or part of the C-terminal globular C1qhomology domain and having activity are selected from amino acids20-333, 109-333, 125-333, 128-333, 140-333, 160-333, 164-333, 179-333,182-333, 185-333, 188-333, 191-333, 193-333, 201-333, 202-333, 227-333,252-333, 252-267, 252-317, 256-267, 256-317, or 304-317 of SEQ ID NO: 2or 4. In other yet more preferred embodiments, said Cluster 1polypeptide fragments having activity are selected from amino acids1-225, 17-225, 20-225, 32-225, 52-225, 56-225, 71-225, 74-225, 77-225,80-225, 83-225, 85-225, 93-225, 94-225, 119-225, 144-225, 144-159,144-209, 148-159, 148-209, or 196-209 of SEQ ID NO: 6. In other yet morepreferred embodiments, said GMG-6A polypeptide fragments having activityare selected from amino acids 20-330, 75-330, 122-330, 125-330, 146-330,157-330, 161-330, 176-330, 179-330, 182-330, 185-330, 188-330, 190-330,198-330, 199-330, 224-330, 249-330, 249-264, 249-314, 253-264, 253-314or 301-314 of SEQ ID NO: 8. In other yet more preferred embodiments,said GMG-6B polypeptide fragments having activity are selected fromamino acids 20-323, 68-323, 115-323, 118-323, 139-323, 150-323, 154-323,169-323, 172-323, 175-323, 178-323, 181-323, 183-323, 191-323, 192-323,217-323, 242-323, 242-257, 242-307, 246-257, 246-307, or 294-307 of SEQID NO: 10.

In yet other preferred embodiments, the invention features a GMG-3,GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide fragment comprising atleast 115, but not more than 175 contiguous amino acids of any one ofthe GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B fragment sequences setforth in the sequence listing, wherein no more than 24 of said at least115 and no more than 175 contiguous amino acids are present in thecollagen-like region of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B.Preferably, the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptidefragment comprises at least 125, but not more than 165, or at least 140,but not more than 165 amino acids, and no more than 24 amino acids arein the collagen-like region; more preferably at least 125 but not morethan 165, or at least 140 but not more than 165 amino acids, and no morethan 12 amino acids are in the collagen-like region; or at least 140 andnot more than 150 amino acids, and no more than 8 amino acids arepresent in the collagen-like region. Preferably the GMG-3, GMG-4,Cluster 1, GMG-6A, or GMG-6B fragment is mammalian, preferably human ormouse, but most preferably human.

GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide fragments of theinvention include variants, fragments, analogs and derivatives of theGMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide fragmentsdescribed above, including modified GMG-3, GMG-4, Cluster 1, GMG-6A, orGMG-6B polypeptide fragments.

Proteolytic cleavage of full-length GMG-3, GMG-4, Cluster 1, GMG-6A, orGMG-6B polypeptides of the invention in vivo is believed to be subjectto regulation that facilitates the appropriate and effective generationof GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide fragments ofthe invention comprised of all or part of the C-terminal globular C1qhomology domain and having lipid partitioning, lipid metabolism, andinsulin-like activity. Said proteolytic cleavage at least in part isregulated at the level of the protease, for example at the level oftissue distribution of the protease and at the level of amount of theprotease, which itself can be regulated by physiological signals such asthose associated with inflammation. Particularly preferred GMG-3, GMG-4,Cluster 1, GMG-6A, or GMG-6B polypeptide fragments of the inventioncomprised of all or part of the C-terminal globular C1q homology domainand having lipid partitioning, lipid metabolism, and insulin-likeactivity are said GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptidefragments of SEQ ID NO: 2, 4, 6, 8, or 10 believed to be generatedproteolytically in vivo.

Said particularly preferred are GMG-3 or GMG-4 polypeptide fragments ofamino acids 45-333, 75-333, or 81-333 of SEQ ID NO: 2 or 4 generated bycollagenase cleavage. Also said particularly preferred are GMG-6Apolypeptide fragments of amino acids 45-330, 72-330, or 78-330 of SEQ IDNO: 8 generated by collagenase cleavage. Also said particularlypreferred are GMG-6B polypeptide fragments of amino acids 65-323 or71-323 of SEQ ID NO: 10 generated by collagenase cleavage. Also saidparticularly preferred are GMG-3 or GMG-4 polypeptide fragments of aminoacids 45-333, 75-333, or 81-333 of SEQ ID NO: 2 or 4 generated by matrixmetalloproteinase-1 (MMP-1) cleavage. Also said particularly preferredare GMG-6A polypeptide fragments of amino acids 45-330, 72-330, or78-330 of SEQ ID NO: 8 generated by matrix metalloproteinase-1 (MMP-1)cleavage. Also said particularly preferred are GMG-6B polypeptidefragments of amino acids 65-323 or 71-323 of SEQ ID NO: 10 generated bymatrix metalloproteinase-1 (MMP-1) cleavage.

Also said particularly preferred are GMG-3 polypeptide fragments ofamino acids 43-333, 46-333, 50-333, 53-333, 61-333, 67-333, 74-333,77-333, 82-333, 86-333, 89-333, 95-333, 100-333, 104-333, 109-333,113-333, 116-333, 125-333, 128-333, 140-333, 160-333, 164-333, 179-333,182-333, 185-333, 188-333, 191-333, 193-333, or 202-333 of SEQ ID NO:2generated by plasmin cleavage. Also said particularly preferred areGMG-4 polypeptide fragments of amino acids 43-333, 46-333, 50-333,53-333, 67-333, 74-333, 77-333, 82-333, 86-333, 89-333, 95-333, 100-333,104-333, 109-333, 113-333, 116-333, 125-333, 128-333, 140-333, 160-333,164-333, 179-333, 182-333, 185-333, 188-333, 191-333, 193-333, or202-333 of SEQ ID NO:4 generated by plasmin cleavage. Also saidparticularly preferred are Cluster 1 polypeptide fragments of aminoacids 5-225, 8-225, 17-225, 20-225, 32-225, 52-225, 56-225, 71-225,74-225, 77-225, 80-225, 83-225, 85-225 or 94-225 of SEQ ID NO: 6generated by plamsin cleavage. Also particularly preferred are GMG-6Apolypeptide fragments of amino acids 43-330, 46-330, 50-333, 53-330,64-330, 68-330, 71-330, 79-330, 83-330, 86-330, 92-330, 97-330, 101-330,122-330, 125-330, 146-330, 157-330, 161-330, 176-330, 179-330, 182-330,185-330, 188-330, 190-330 or 199-330 of SEQ ID NO: 8 generated byplasmin cleavage. Also said particularly preferred are GMG-6Bpolypeptide fragments of amino acids 43-323, 46-323, 57-323, 61-323,64-323, 72-323, 76-323, 79-323, 85-323, 90-323, 94-323, 115-323,118-323, 139-323, 150-323, 154-323, 169-323, 172-323, 175-323, 178-323,181-323, 183-323 or 192-323 of SEQ ID NO: 10 generated by plasmincleavage.

GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides of the inventioninclude variants, fragments, analogs and derivatives of the GMG-3,GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides described above,including modified GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bpolypeptides.

Variants

It will be recognized by one of ordinary skill in the art that someamino acids of the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bpolypeptide sequences of the present invention can be varied withoutsignificant effect on the structure or function of the proteins; therewill be critical amino acids in the sequence that determine activity.Thus, the invention further includes variants of GMG-3, GMG-4, Cluster1, GMG-6A, or GMG-6B polypeptides that have metabolic-related activityas described above. Such variants include GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B polypeptide sequences with one or more amino aciddeletions, insertions, inversions, repeats, and substitutions eitherfrom natural mutations or human manipulation selected according togeneral rules known in the art so as to have little effect on activity.Guidance concerning how to make phenotypically silent amino acidsubstitutions is provided below.

There are two main approaches for studying the tolerance of an aminoacid sequence to change (see, Bowie, et al. (1990) Science, 247,1306-10). The first method relies on the process of evolution, in whichmutations are either accepted or rejected by natural selection. Thesecond approach uses genetic engineering to introduce amino acid changesat specific positions of a cloned gene and selections or screens toidentify sequences that maintain functionality.

These studies have revealed that proteins are surprisingly tolerant ofamino acid substitutions and indicate which amino acid changes arelikely to be permissive at a certain position of the protein. Forexample, most buried amino acid residues require nonpolar side chains,whereas few features of surface side chains are generally conserved.Other such phenotypically silent substitutions are described by Bowie etal. (supra) and the references cited therein.

In the case of an amino acid substitution in the amino acid sequence ofa polypeptide according to the invention, one or several amino acids canbe replaced by “equivalent” amino acids. The expression “equivalent”amino acid is used herein to designate any amino acid that may besubstituted for one of the amino acids having similar properties, suchthat one skilled in the art of peptide chemistry would expect thesecondary structure and hydropathic nature of the polypeptide to besubstantially unchanged.

In particular embodiments, conservative substitutions of interest areshown in Table 1 under the heading of preferred substitutions. If suchsubstitutions result in a change in biological activity, then moresubstantial changes, denominated exemplary substitutions in Table 1, oras further described below in reference to amino acid classes, areintroduced and the products screened.

TABLE 1 Original Residue Exemplary Substitutions Preferred SubstitutionsAla (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his;lys; arg gln Asp (D) glu glu Cys (C) ser ser Gln (Q) asn asn Glu (E) aspasp Gly (G) pro; ala ala His (H) asn; gln; lys; arg arg Ile (I) leu;val; met; ala; phe; norleucine leu Leu (L) norleucine; ile; val; met;ala; phe ile Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe (F)leu; val; ile; ala; tyr leu Pro (P) ala ala Ser (S) thr thr Thr (T) serser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile;leu; met; phe; ala; norleucine leu

Substantial modifications in function or immunological identity of theGMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides are accomplishedby selecting substitutions that differ significantly in their effect onmaintaining (a) the structure of the polypeptide backbone in the area ofthe substitution, for example, as a sheet or helical conformation, (b)the charge or hydrophobicity of the molecule at the target site, or (c)the bulk of the side chain. Naturally occurring residues are dividedinto groups based on common side-chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gln, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Such substituted residues also may beintroduced into the conservative substitution sites or, more preferably,into the remaining (non-conserved) sites.

The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis [Carter et al., NuclAcids Res, 13:4331 (1986); Zoller et al., Nucl Acids Res, 10:6487(1987)], cassette mutagenesis [Wells et al. Gene, 34:315 (1985)],restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc.London SerA, 317:415 (1986)] or other known techniques can be performedon the cloned DNA to produce the GMG-3, GMG-4, Cluster 1, GMG-6A, orGMG-6B variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence. Among the preferredscanning amino acids are relatively small, neutral amino acids. Suchamino acids include alanine, glycine, serine, and cysteine. Alanine istypically a preferred scanning amino acid among this group because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main chain conformation of the variant [Cunningham and Wells,Science, 244: 1081-1085 (1989)]. Alanine is also typically preferredbecause it is the most common amino acid. Further, it is frequentlyfound in both buried and exposed positions [Creighton, The Proteins,(W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)]. Ifalanine substitution does not yield adequate amounts of variant, anisoteric amino acid can be used.

Amino acids in the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bpolypeptide sequences of the invention that are essential for functioncan also be identified by methods known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (see, e.g.,Cunningham, et al. (1989) Science 244:1081-5). The latter procedureintroduces single alanine mutations at every residue in the molecule.The resulting mutant molecules are then tested for metabolic-relatedactivity using assays as described above. Of special interest aresubstitutions of charged amino acids with other charged or neutral aminoacids that may produce proteins with highly desirable improvedcharacteristics, such as less aggregation. Aggregation may not onlyreduce activity but also be problematic when preparing pharmaceutical orphysiologically acceptable formulations, because aggregates can beimmunogenic (see, e.g., Pinckard, et al., (1967) Clin Exp Immunol2:331-340; Robbins, et al., (1987) Diabetes 36:838-41; and Cleland, etal., (1993) Crit Rev Ther Drug Carrier Syst 10:307-77).

Thus, the fragment, derivative, analog, or homolog of the GMG-3, GMG-4,Cluster 1, GMG-6A, or GMG-6B polypeptides of the present invention maybe, for example: (i) one in which one or more of the amino acid residuesare substituted with a conserved or non-conserved amino acid residue(preferably a conserved amino acid residue) and such substituted aminoacid residue may or may not be one encoded by the genetic code (i.e. maybe a non-naturally occurring amino acid); or (ii) one in which one ormore of the amino acid residues includes a substituent group; or (iii)one in which the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptidesare fused with another compound, such as a compound to increase thehalf-life of the fragment (for example, polyethylene glycol); or (iv)one in which the additional amino acids are fused to the above form ofthe fragment, such as an IgG Fc fusion region peptide or leader orsecretory sequence or a sequence which is employed for purification ofthe above form of the fragment or a pro-protein sequence. Suchfragments, derivatives and analogs are deemed to be within the scope ofthose skilled in the art from the teachings herein.

A further embodiment of the invention relates to a polypeptide whichcomprises the amino acid sequence of GMG-3, GMG-4, Cluster 1, GMG-6A, orGMG-6B polypeptides having an amino acid sequence which contains atleast one conservative amino acid substitution, but not more than 50conservative amino acid substitutions, not more than 40 conservativeamino acid substitutions, not more than 30 conservative amino acidsubstitutions, and not more than 20 conservative amino acidsubstitutions. Also provided are polypeptides which comprise the aminoacid sequence of a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B fragment,having at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1conservative amino acid substitutions.

In addition, amino acids have chirality within the body of either L orD. In some embodiments it is preferable to alter the chirality of theamino acids in the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bpolypeptide fragments of the invention in order to extend half-lifewithin the body. Thus, in some embodiments, one or more of the aminoacids are preferably in the L configuration. In other embodiments, oneor more of the amino acids are preferably in the D configuration.

Percent Identity

The polypeptides of the present invention also include polypeptideshaving an amino acid sequence at least 50% identical, at least 60%identical, or 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99% identical to a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bpolypeptide as described above. By a polypeptide having an amino acidsequence at least, for example, 95% “identical” to a GMG-3, GMG-4,Cluster 1, GMG-6A, or GMG-6B polypeptide amino acid sequence is meantthat the amino acid sequence is identical to the GMG-3, GMG-4, Cluster1, GMG-6A, or GMG-6B polypeptide sequence except that it may include upto five amino acid alterations per each 100 amino acids of the GMG-3,GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide amino acid sequence. Thereference sequence is the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bpolypeptide with a sequence corresponding to the sequences provided inSEQ ID NOs: 2, 4, 6, 8, or 10. Thus, to obtain a polypeptide having anamino acid sequence at least 95% identical to a GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B polypeptide amino acid sequence, up to 5% (5 of 100)of the amino acid residues in the sequence may be inserted, deleted, orsubstituted with another amino acid compared with the GMG-3, GMG-4,Cluster 1, GMG-6A, or GMG-6B polypeptide sequence. These alterations mayoccur at the amino or carboxy termini or anywhere between those terminalpositions, interspersed either individually among residues in thesequence or in one or more contiguous groups within the sequence.

As a practical matter, whether any particular polypeptide is apercentage identical to a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bpolypeptide can be determined conventionally using known computerprograms. Such algorithms and programs include, but are by no meanslimited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson andLipman, (1988) Proc Natl Acad Sci USA 85:2444-8; Altschul et al., (1990)J Mol Biol 215:403-410; Thompson et al., (1994) Nucleic Acids Res22(2):4673-4680; Higgins et al, (1996) Meth Enzymol 266:383-402;Altschul et al, (1997) Nucleic Acids Res 25:3389-3402; Altschul et al.,(1993) Nature Genetics 3:266-272). In a particularly preferredembodiment, protein and nucleic acid sequence homologies are evaluatedusing the Basic Local Alignment Search Tool (“BLAST”), which is wellknown in the art (See, e.g., Karlin and Altschul (1990) Proc Natl AcadSci USA 87:2264-8; Altschul et al., 1990, 1993, 1997, all supra). Inparticular, five specific BLAST programs are used to perform thefollowing tasks:

(1) BLASTP and BLAST3 compare an amino acid query sequence against aprotein sequence database;

(2) BLASTN compares a nucleotide query sequence against a nucleotidesequence database;

(3) BLASTX compares the six-frame conceptual translation products of aquery nucleotide sequence (both strands) against a protein sequencedatabase;

(4) TBLASTN compares a query protein sequence against a nucleotidesequence database translated in all six reading frames (both strands);and

(5) TBLASTX compares the six-frame translations of a nucleotide querysequence against the six-frame translations of a nucleotide sequencedatabase.

The BLAST programs identify homologous sequences by identifying similarsegments, which are referred to herein as “high-scoring segment pairs,”between a query amino or nucleic acid sequence and a test sequence whichis preferably obtained from a protein or nucleic acid sequence database.High-scoring segment pairs are preferably identified (i.e., aligned) bymeans of a scoring matrix, many of which are known in the art.Preferably, the scoring matrix used is the BLOSUM62 matrix (see, Gonnetet al., (1992) Science 256:1443-5; Henikoff and Henikoff (1993) Proteins17:49-61). Less preferably, the PAM or PAM250 matrices may also be used(See, e.g., Schwartz and Dayhoff, eds, (1978) Matrices for DetectingDistance Relationships: Atlas of Protein Sequence and Structure,Washington: National Biomedical Research Foundation). The BLAST programsevaluate the statistical significance of all high-scoring segment pairsidentified, and preferably selects those segments which satisfy auser-specified threshold of significance, such as a user-specifiedpercent homology. Preferably, the statistical significance of ahigh-scoring segment pair is evaluated using the statisticalsignificance formula of Karlin. (See, e.g., Karlin and Altschul, (1990)Proc Natl Acad Sci USA 87:2264-8). The BLAST programs may be used withthe default parameters or with modified parameters provided by the user.Preferably, the parameters are default parameters.

A preferred method for determining the best overall match between aquery sequence (a sequence of the present invention) and a subjectsequence, also referred to as a global sequence alignment, can bedetermined using the FASTDB computer program based on the algorithm ofBrutlag et al. (1990) Comp App Biosci 6:237-245. In a sequence alignmentthe query and subject sequences are both amino acid sequences. Theresult of said global sequence alignment is in percent identity.Preferred parameters used in a FASTDB amino acid alignment are:Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20,Randomization Group=25 Length=0, Cutoff Score=1, Window Size=sequencelength, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=247 or thelength of the subject amino acid sequence, whichever is shorter.

If the subject sequence is shorter than the query sequence due to N- orC-terminal deletions, not because of internal deletions, the results, inpercent identity, must be manually corrected because the FASTDB programdoes not account for N- and C-terminal truncations of the subjectsequence when calculating global percent identity. For subject sequencestruncated at the N- and C-termini, relative to the query sequence, thepercent identity is corrected by calculating the number of residues ofthe query sequence that are N- and C-terminal of the subject sequence,that are not matched/aligned with a corresponding subject residue, as apercent of the total bases of the query sequence. Whether a residue ismatched/aligned is determined by results of the FASTDB sequencealignment. This percentage is then subtracted from the percent identity,calculated by the above FASTDB program using the specified parameters,to arrive at a final percent identity score. This final percent identityscore is what is used for the purposes of the present invention. Onlyresidues to the N- and C-termini of the subject sequence, which are notmatched/aligned with the query sequence, are considered for the purposesof manually adjusting the percent identity score. That is, only queryamino acid residues outside the farthest N- and C-terminal residues ofthe subject sequence.

For example, a 90 amino acid residue subject sequence is aligned with a100-residue query sequence to determine percent identity. The deletionoccurs at the N-terminus of the subject sequence and therefore, theFASTDB alignment does not match/align with the first residues at theN-terminus. The 10 unpaired residues represent 10% of the sequence(number of residues at the N- and C-termini not matched/total number ofresidues in the query sequence) so 10% is subtracted from the percentidentity score calculated by the FASTDB program. If the remaining 90residues were perfectly matched the final percent identity would be 90%.

In another example, a 90-residue subject sequence is compared with a100-residue query sequence. This time the deletions are internal sothere are no residues at the N- or C-termini of the subject sequence,which are not matched/aligned with the query. In this case, the percentidentity calculated by FASTDB is not manually corrected. Once again,only residue positions outside the N- and C-terminal ends of the subjectsequence, as displayed in the FASTDB alignment, which are notmatched/aligned with the query sequence are manually corrected. No othermanual corrections are made for the purposes of the present invention.

Production

Note, throughout the disclosure, wherever GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B polypeptides are discussed, GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B fragments, variants and derivatives are specificallyintended to be included as a preferred subset of GMG-3, GMG-4, Cluster1, GMG-6A, or GMG-6B polypeptides.

GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides are preferablyisolated from human or mammalian tissue samples or expressed from humanor mammalian genes in human or mammalian cells. The GMG-3, GMG-4,Cluster 1, GMG-6A, or GMG-6B polypeptides of the invention can be madeusing routine expression methods known in the art. The polynucleotideencoding the desired polypeptide is ligated into an expression vectorsuitable for any convenient host. Both eukaryotic and prokaryotic hostsystems are used in forming recombinant polypeptides. The polypeptide isthen isolated from lysed cells or from the culture medium and purifiedto the extent needed for its intended use. Purification is by anytechnique known in the art, for example, differential extraction, saltfractionation, chromatography, centrifugation, and the like. See, forexample, Methods in Enzymology for a variety of methods for purifyingproteins.

In a alternative embodiment, the polypeptides of the invention areisolated from milk. The polypeptides can be purified as full lengthGMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides, which can thenbe cleaved, if appropriate, in vitro to generate a GMG-3, GMG-4, Cluster1, GMG-6A, or GMG-6B fragment, or, alternatively, GMG-3, GMG-4, Cluster1, GMG-6A, or GMG-6B fragments themselves can be purified from the milk.Any of a large number of methods can be used to purify the presentpolypeptides from milk including those taught in Protein PurificationApplications, A Practical Approach (New Edition), Edited by Simon Roe,AEA Technology Products and Systems, Biosciences, Harwell; Clark (1998)J Mammary Gland Biol Neoplasia 3:337-50; Wilkins and Velander (1992)49:333-8; U.S. Pat. Nos. 6,140,552; 6,025,540; Hennighausen, ProteinExpression and Purification, vol. 1, pp. 3-8 (1990); Harris et al.(1997) Bioseparation 7:31-7; Degener et al. (1998) J Chromatog799:125-37; Wilkins (1993) J Cell Biochem. Suppl. 0 (17 part A):39; theentire disclosures of each of which are herein incorporated byreference. In a typical embodiment, milk is centrifuged, e.g. at arelatively low speed, to separate the lipid fraction, and the aqueoussupernatant is then centrifuged at a higher speed to separate the caseinin the milk from the remaining, “whey” fraction. Often, biomedicalproteins are found in this whey fraction, and can be isolated from thisfraction using standard chromatographic or other procedures commonlyused for protein purification, e.g. as described elsewhere in thepresent application. In one preferred embodiment, GMG-3, GMG-4, Cluster1, GMG-6A, or GMG-6B polypeptides are purified using antibodies specificto GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides, e.g. usingaffinity chromatography. In addition, methods can be used to isolateparticular GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B fragments, e.g.electrophoretic or other methods for isolating proteins of a particularsize. The GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptidesisolating using these methods can be naturally occurring, as GMG-3,GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides have been discovered tobe naturally present in the milk of mammals, or can be the result of therecombinant production of the protein in the mammary glands of anon-human mammal, as described infra. In one such embodiment, the GMG-3,GMG-4, Cluster 1, GMG-6A, or GMG-6B is produced as a fusion protein witha heterologous, antigenic polypeptide sequence, which antigenic sequencecan be used to purify the protein, e.g., using standard immuno-affinitymethodology.

In addition, shorter protein fragments may be produced by chemicalsynthesis. Alternatively, the proteins of the invention are extractedfrom cells or tissues of humans or non-human animals. Methods forpurifying proteins are known in the art, and include the use ofdetergents or chaotropic agents to disrupt particles followed bydifferential extraction and separation of the polypeptides by ionexchange chromatography, affinity chromatography, sedimentationaccording to density, and gel electrophoresis.

Any GMG-3, GMG-4, GMG-6A, or GMG-6B cDNA or Cluster 1 polynucleotide,including those in SEQ ID NO: 1, 3, 5, 7, or 9, can be used to expressGMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides. The nucleicacid encoding the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B to beexpressed is operably linked to a promoter in an expression vector usingconventional cloning technology. The GMG-3, GMG-4, GMG-6A, or GMG-6BcDNA or Cluster 1 polynucleotide insert in the expression vector maycomprise the coding sequence for: the full-length GMG-3, GMG-4, Cluster1, GMG-6A, or GMG-6B polypeptide (to be later modified); from 6 aminoacids to 6 amino acids any integer less than the full-length GMG-3,GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide; a GMG-3, GMG-4, Cluster1, GMG-6A, or GMG-6B fragment; or variants and % similar polypeptides.

The expression vector is any of the mammalian, yeast, insect orbacterial expression systems known in the art, some of which aredescribed herein. Commercially available vectors and expression systemsare available from a variety of suppliers including Genetics Institute(Cambridge, Mass.), Stratagene (La Jolla, Calif.), Promega (Madison,Wis.), and Invitrogen (San Diego, Calif.). If desired, to enhanceexpression and facilitate proper protein folding, the codon context andcodon pairing of the sequence can be optimized for the particularexpression organism into which the expression vector is introduced, asexplained by Hatfield, et al., U.S. Pat. No. 5,082,767, the disclosuresof which are incorporated by reference herein in their entirety.

If the nucleic acid encoding any one of the GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B polypeptides lacks a methionine to serve as theinitiation site, an initiating methionine can be introduced next to thefirst codon of the nucleic acid using conventional techniques.Similarly, if the insert from the GMG-3, GMG-4, Cluster 1, GMG-6A, orGMG-6B polypeptide cDNA lacks a poly A signal, this sequence can beadded to the construct by, for example, splicing out the Poly A signalfrom pSG5 (Stratagene) using BglI and SalI restriction endonucleaseenzymes and incorporating it into the mammalian expression vector pXT1(Stratagene). pXT1 contains the LTRs and a portion of the gag gene fromMoloney Murine Leukemia Virus. The position of the LTRs in the constructallows efficient stable transfection. The vector includes the HerpesSimplex Thymidine Kinase promoter and the selectable neomycin gene.

The nucleic acid encoding GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B canbe obtained by PCR from a vector containing the GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B nucleotide sequence using oligonucleotide primerscomplementary to the desired GMG-3, GMG-4, GMG-6A, or GMG-6B cDNA orCluster 1 polynucleotide and containing restriction endonucleasesequences for Pst I incorporated into the 5′ primer and BglII at the 5′end of the corresponding cDNA 3′ primer, taking care to ensure that thesequence encoding the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B ispositioned properly with respect to the poly A signal. The purifiedpolynucleotide obtained from the resulting PCR reaction is digested withPstI, blunt ended with an exonuclease, digested with Bgl II, purifiedand ligated to pXT1, now containing a poly A signal and digested withBglII.

Transfection of a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B expressingvector into mouse NIH 3T3 cells is one embodiment of introducingpolynucleotides into host cells. Introduction of a polynucleotideencoding a polypeptide into a host cell can be effected by calciumphosphate transfection, DEAE-dextran mediated transfection, cationiclipid-mediated transfection, electroporation, transduction, infection,or other methods. Such methods are described in many standard laboratorymanuals, such as Davis et al. ((1986) Methods in Molecular Biology,Elsevier Science Publishing Co., Inc., Amsterdam). It is specificallycontemplated that the polypeptides of the present invention may in factbe expressed by a host cell lacking a recombinant vector.

A polypeptide of this invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography (“HPLC”) is employed for purification.Polypeptides of the present invention, and preferably the secreted form,can also be recovered from: products purified from natural sources,including bodily fluids, tissues and cells, whether directly isolated orcultured; products of chemical synthetic procedures; and productsproduced by recombinant techniques from a prokaryotic or eukaryotichost, including, for example, bacterial, yeast, higher plant, insect,and mammalian cells.

Depending upon the host employed in a recombinant production procedure,the polypeptides of the present invention may be glycosylated or may benon-glycosylated. Preferably the polypeptides of the invention arenon-glycosylated. In addition, polypeptides of the invention may alsoinclude an initial modified methionine residue, in some cases as aresult of host-mediated processes. Thus, it is well known in the artthat the N-terminal methionine encoded by the translation initiationcodon generally is removed with high efficiency from any protein aftertranslation in all eukaryotic cells. While the N-terminal methionine onmost proteins also is efficiently removed in most prokaryotes, for someproteins, this prokaryotic removal process is inefficient, depending onthe nature of the amino acid to which the N-terminal methionine iscovalently linked.

In addition to encompassing host cells containing the vector constructsdiscussed herein, the invention also encompasses primary, secondary, andimmortalized host cells of vertebrate origin, particularly mammalianorigin, that have been engineered to delete or replace endogenousgenetic material (e.g., coding sequence), and/or to include geneticmaterial (e.g., heterologous polynucleotide sequences) that is operablyassociated with the polynucleotides of the invention, and whichactivates, alters, and/or amplifies endogenous polynucleotides. Forexample, techniques known in the art may be used to operably associateheterologous control regions (e.g., promoter and/or enhancer) andendogenous polynucleotide sequences via homologous recombination, see,e.g. U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; InternationalPublication No. WO 96/29411, published Sep. 26, 1996; InternationalPublication No. WO 94/12650, published Aug. 4, 1994; Koller et al.,(1989) Proc Natl Acad Sci USA 86:8932-5; Koller et al., (1989) Proc NatlAcad Sci USA 86:8927-31; and Zijlstra et al. (1989) Nature 342:435-8;the disclosures of each of which are incorporated by reference in theirentireties).

Modifications

In addition, polypeptides of the invention can be chemically synthesizedusing techniques known in the art (See, e.g., Creighton, 1983 Proteins.New York, N.Y.: W.H. Freeman and Company; and Hunkapiller et al., (1984)Nature 310:105-11). For example, a relative short fragment of theinvention can be synthesized by use of a peptide synthesizer.Furthermore, if desired, nonclassical amino acids or chemical amino acidanalogs can be introduced as a substitution or addition into thefragment sequence. Non-classical amino acids include, but are notlimited to, to the D-isomers of the common amino acids,2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid,Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib,2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine,norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline,cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,cyclohexylalanine, b-alanine, fluoroamino acids, designer amino acidssuch as b-methyl amino acids, Ca-methyl amino acids, Na-methyl aminoacids, and amino acid analogs in general. Furthermore, the amino acidcan be D (dextrorotary) or L (levorotary).

The invention encompasses polypeptides which are differentially modifiedduring or after translation, e.g., by glycosylation, acetylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to an antibody molecule or othercellular ligand, etc. Any of numerous chemical modifications may becarried out by known techniques, including but not limited to, specificchemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8protease, NaBH4; acetylation, formylation, oxidation, reduction;metabolic synthesis in the presence of tunicamycin; etc.

Additional post-translational modifications encompassed by the inventioninclude, for example, N-linked or O-linked carbohydrate chains,processing of N-terminal or C-terminal ends), attachment of chemicalmoieties to the amino acid backbone, chemical modifications of N-linkedor O-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of procaryotic host cellexpression. The polypeptides may also be modified with a detectablelabel, such as an enzymatic, fluorescent, isotopic or affinity label toallow for detection and isolation of the polypeptide.

Also provided by the invention are chemically modified derivatives ofthe polypeptides of the invention that may provide additional advantagessuch as increased solubility, stability and circulating time of thepolypeptide, or decreased immunogenicity. See U.S. Pat. No. 4,179,337.The chemical moieties for derivitization may be selected from watersoluble polymers such as polyethylene glycol, ethylene glycol/propyleneglycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcoholand the like. The polypeptides may be modified at random positionswithin the molecule, or at predetermined positions within the moleculeand may include one, two, three or more attached chemical moieties.

The polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weight isbetween about 1 kDa and about 100 kDa (the term “about” indicating thatin preparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog).

The polyethylene glycol molecules (or other chemical moieties) should beattached to the polypeptide with consideration of effects on functionalor antigenic domains of the polypeptide. There are a number ofattachment methods available to those skilled in the art, e.g., EP 0 401384, herein incorporated by reference (coupling PEG to G-CSF), see alsoMalik et al. (1992) Exp Hematol (8):1028-35, reporting pegylation ofGM-CSF using tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas, a free amino or carboxyl group. Reactive groups are those to whichan activated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group may include lysine residues and theN-terminal amino acid residues; those having a free carboxyl group mayinclude aspartic acid residues, glutamic acid residues and theC-terminal amino acid residue. Sulfhydryl groups may also be used as areactive group for attaching the polyethylene glycol molecules.Preferred for therapeutic purposes is attachment at an amino group, suchas attachment at the N-terminus or lysine group.

One may specifically desire proteins chemically modified at theN-terminus. Using polyethylene glycol as an illustration of the presentcomposition, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (polypeptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective proteins chemicallymodified at the N-terminus may be accomplished by reductive alkylation,which exploits differential reactivity of different types of primaryamino groups (lysine versus the N-terminal) available for derivatizationin a particular protein. Under the appropriate reaction conditions,substantially selective derivatization of the protein at the N-terminuswith a carbonyl group containing polymer is achieved.

Multimers

The polypeptides of the invention may be in monomers or multimers (i.e.,dimers, trimers, tetramers and higher multimers). Accordingly, thepresent invention relates to monomers and multimers of the polypeptidesof the invention, their preparation, and compositions (preferably,pharmaceutical or physiologically acceptable compositions) containingthem. In specific embodiments, the polypeptides of the invention aremonomers, dimers, trimers or tetramers. In additional embodiments, themultimers of the invention are at least dimers, at least trimers, or atleast tetramers.

Multimers encompassed by the invention may be homomers or heteromers. Asused herein, the term homomer, refers to a multimer containing onlypolypeptides corresponding to the GMG-3, GMG-4, Cluster 1, GMG-6A, orGMG-6B polypeptides of the invention (including polypeptide fragments,variants, splice variants, and fusion proteins corresponding to thesepolypeptide fragments as described herein). These homomers may containpolypeptide fragments having identical or different amino acidsequences. In a specific embodiment, a homomer of the invention is amultimer containing only polypeptide fragments having an identical aminoacid sequence. In another specific embodiment, a homomer of theinvention is a multimer containing polypeptide fragments havingdifferent amino acid sequences. In specific embodiments, the multimer ofthe invention is a homodimer (e.g., containing polypeptides havingidentical or different amino acid sequences) or a homotrimer (e.g.,containing polypeptides having identical and/or different amino acidsequences). In additional embodiments, the homomeric multimer of theinvention is at least a homodimer, at least a homotrimer, or at least ahomotetramer.

As used herein, the term heteromer refers to a multimer containing oneor more heterologous polypeptides (i.e., corresponding to differentproteins or polypeptides thereof) in addition to the polypeptides of theinvention. In a specific embodiment, the multimer of the invention is aheterodimer, a heterotrimer, or a heterotetramer. In additionalembodiments, the heteromeric multimer of the invention is at least aheterodimer, at least a heterotrimer, or at least a heterotetramer.

Multimers of the invention may be the result of hydrophobic,hydrophilic, ionic and/or covalent associations and/or may be indirectlylinked, by for example, liposome formation. Thus, in one embodiment,multimers of the invention, such as, for example, homodimers orhomotrimers, are formed when polypeptides of the invention contact oneanother in solution. In another embodiment, heteromultimers of theinvention, such as, for example, heterotrimers or heterotetramers, areformed when polypeptides of the invention contact antibodies to thepolypeptides of the invention (including antibodies to the heterologouspolypeptide sequence in a fusion protein of the invention) in solution.In other embodiments, multimers of the invention are formed by covalentassociations with and/or between the polypeptides of the invention. Suchcovalent associations may involve one or more amino acid residuescontained in the polypeptide sequence (e.g., that recited in thesequence listing, or contained in the polypeptide encoded by a depositedclone). In one instance, the covalent associations are cross-linkingbetween cysteine residues located within the polypeptide sequences,which interact in the native (i.e., naturally occurring) polypeptide. Inanother instance, the covalent associations are the consequence ofchemical or recombinant manipulation. Alternatively, such covalentassociations may involve one or more amino acid residues contained inthe heterologous polypeptide sequence in a fusion protein of theinvention.

In one example, covalent associations are between the heterologoussequence contained in a fusion protein of the invention (see, e.g., U.S.Pat. No. 5,478,925). In a specific example, the covalent associationsare between the heterologous sequence contained in an Fc fusion proteinof the invention (as described herein). In another specific example,covalent associations of fusion proteins of the invention are betweenheterologous polypeptide sequence from another protein that is capableof forming covalently associated multimers, such as for example,oseteoprotegerin (see, e.g., International Publication NO: WO 98/49305,the contents of which are herein incorporated by reference in itsentirety). In another embodiment, two or more polypeptides of theinvention are joined through peptide linkers. Examples include thosepeptide linkers described in U.S. Pat. No. 5,073,627 (herebyincorporated by reference). Proteins comprising multiple polypeptides ofthe invention separated by peptide linkers may be produced usingconventional recombinant DNA technology.

Another method for preparing multimer polypeptides of the inventioninvolves use of polypeptides of the invention fused to a leucine zipperor isoleucine zipper polypeptide sequence. Leucine zipper and isoleucinezipper domains are polypeptides that promote multimerization of theproteins in which they are found. Leucine zippers were originallyidentified in several DNA-binding proteins, and have since been found ina variety of different proteins (Landschulz et al., (1988) Genes Dev2:786-800). Among the known leucine zippers are naturally occurringpeptides and derivatives thereof that dimerize or trimerize. Examples ofleucine zipper domains suitable for producing soluble multimericproteins of the invention are those described in PCT application WO94/10308, hereby incorporated by reference. Recombinant fusion proteinscomprising a polypeptide of the invention fused to a polypeptidesequence that dimerizes or trimerizes in solution are expressed insuitable host cells, and the resulting soluble multimeric fusion proteinis recovered from the culture supernatant using techniques known in theart.

Trimeric polypeptides of the invention may offer the advantage ofenhanced biological activity. Preferred leucine zipper moieties andisoleucine moieties are those that preferentially form trimers. Oneexample is a leucine zipper derived from lung surfactant protein D(SPD), as described in Hoppe et al. FEBS Letters (1994) 344:191-5 and inU.S. patent application Ser. No. 08/446,922, hereby incorporated byreference. Other peptides derived from naturally occurring trimericproteins may be employed in preparing trimeric polypeptides of theinvention. In another example, proteins of the invention are associatedby interactions between Flag® & polypeptide sequence contained in fusionproteins of the invention containing Flag® polypeptide sequence. In afurther embodiment, proteins of the invention are associated byinteractions between heterologous polypeptide sequence contained inFlag® fusion proteins of the invention and anti Flag® antibody.

The multimers of the invention may be generated using chemicaltechniques known in the art. For example, polypeptides desired to becontained in the multimers of the invention may be chemicallycross-linked using linker molecules and linker molecule lengthoptimization techniques known in the art (see, e.g., U.S. Pat. No.5,478,925, which is herein incorporated by reference in its entirety).Additionally, multimers of the invention may be generated usingtechniques known in the art to form one or more inter-moleculecross-links between the cysteine residues located within the sequence ofthe polypeptides desired to be contained in the multimer (see, e.g.,U.S. Pat. No. 5,478,925, which is herein incorporated by reference inits entirety). Further, polypeptides of the invention may be routinelymodified by the addition of cysteine or biotin to the C-terminus orN-terminus of the polypeptide and techniques known in the art may beapplied to generate multimers containing one or more of these modifiedpolypeptides (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety). Additionally, at least 30techniques known in the art may be applied to generate liposomescontaining the polypeptide components desired to be contained in themultimer of the invention (see, e.g., U.S. Pat. No. 5,478,925, which isherein incorporated by reference in its entirety).

Alternatively, multimers of the invention may be generated using geneticengineering techniques known in the art. In one embodiment, polypeptidescontained in multimers of the invention are produced recombinantly usingfusion protein technology described herein or otherwise known in the art(see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated byreference in its entirety). In a specific embodiment, polynucleotidescoding for a homodimer of the invention are generated by ligating apolynucleotide sequence encoding a polypeptide of the invention to asequence encoding a linker polypeptide and then further to a syntheticpolynucleotide encoding the translated product of the polypeptide in thereverse orientation from the original C-terminus to the N-terminus(lacking the leader sequence) (see, e.g., U.S. Pat. No. 5,478,925, whichis herein incorporated by reference in its entirety). In anotherembodiment, recombinant techniques described herein or otherwise knownin the art are applied to generate recombinant polypeptides of theinvention which contain a transmembrane domain (or hyrophobic or signalpeptide) and which can be incorporated by membrane reconstitutiontechniques into liposomes (See, e.g., U.S. Pat. No. 5,478,925, which isherein incorporated by reference in its entirety).

II. GMG-3, GMG-4, Cluster 1, GMG-6A or GMG-6B Polynucleotides of theInvention

Preferred polynucleotides are those that encode GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B polypeptides of the invention. The recombinantpolynucleotides encoding GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bpolypeptides can be used in a variety of ways, including, but notlimited to, expressing the polypeptides in recombinant cells for use inscreening assays for antagonists and agonists of its activity as well asto facilitate its purification for use in a variety of ways including,but not limited to screening assays for agonists and antagonists of itsactivity, diagnostic screens, and raising antibodies, as well astreatment and/or prevention of metabolic-related diseases and disordersand/or to reduce body mass.

The invention relates to the polynucleotides encoding GMG-3, GMG-4,Cluster 1, GMG-6A, or GMG-6B polypeptides and variant polypeptidesthereof as described herein. These polynucleotides may be purified,isolated, and/or recombinant. In all cases, the desired GMG-3, GMG-4,Cluster 1, GMG-6A, or GMG-6B polynucleotides of the invention are thosethat encode GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides ofthe invention having metabolic-related activity as described anddiscussed herein.

Fragments

A polynucleotide fragment is a polynucleotide having a sequence thatentirely is the same as part, but not all, of the full-length GMG-3,GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide or a specified GMG-3,GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide nucleotide sequence.Such fragments may be “free-standing”, i.e. not part of or fused toother polynucleotides, or they may be comprised within anothernon-GMG-3, -GMG-4, -Cluster 1, -GMG-6A, or -GMG-6B (heterologous)polynucleotide of which they form a part or region. However, severalGMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polynucleotide fragments maybe comprised within a single polynucleotide.

The GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polynucleotides of theinvention comprise from 18 consecutive bases to the full-lengthpolynucleotide sequences encoding the intact GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B polypeptides, for example the full-length GMG-3,GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide polynucleotide sequencesin SEQ ID NO: 1, 3, 5, 7, or 9. In one aspect of this embodiment, thepolynucleotide comprises at least 18, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135,140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205,210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275,280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345,350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415,420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485,490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555,560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625,630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695,700, 705, 710, 715, 720, 725, 740, 770, 800, 850, 879, 900, 950, 972,993, 1000 or 1002 consecutive nucleotides of a polynucleotide of thepresent invention.

In addition to the above preferred nucleic acid sizes, further preferrednucleic acids comprise at least 18 nucleotides, wherein “at least 18” isdefined as any integer between 18 and the integer representing the 3′most nucleotide position of the intact GMG-3, GMG-4, GMG-6A, or GMG-6Bpolypeptides cDNA or Cluster 1 polynucleotide as set forth in SEQ IDNOs: 1, 3, 5, 7, or 9 or elsewhere herein.

Further included as preferred polynucleotides of the present inventionare nucleic acid fragments at least 18 nucleotides in length, asdescribed above, that are further specified in terms of their 5′ and 3′position. The 5′ and 3′ positions are represented by the positionnumbers set forth in the sequence listing below. For allelic anddegenerate and other variants, position I is defined as the 5′ mostnucleotide of the ORF, i.e., the nucleotide “A” of the start codon (ATG)with the remaining nucleotides numbered consecutively. Therefore, everycombination of a 5′ and 3′ nucleotide position that a polynucleotidefragment, at least 18 contiguous nucleotides in length, could occupy onan intact GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptideencoding a polynucleotide of the present invention is included in theinvention as an individual species. The polynucleotide fragmentsspecified by 5′ and 3′ positions can be immediately envisaged and aretherefore not individually listed solely for the purpose of notunnecessarily lengthening the specification.

It is noted that the above species of polynucleotide fragments of thepresent invention may alternatively be described by the formula “x toy”; where “x” equals the 5′ most nucleotide position and “y” equals the3′ most nucleotide position of the polynucleotide; and further where “x”equals an integer between 1 and the number of nucleotides of thepolynucleotide sequence of the present invention minus 18, and where “y”equals an integer between 19 and the number of nucleotides of thepolynucleotide sequence of the present invention minus 18 nucleotides;and where “x” is an integer less than “y” by at least 18.

The present invention also provides for the exclusion of any species ofpolynucleotide fragments of the present invention specified by 5′ and 3′positions or polynucleotides specified by size in nucleotides asdescribed above. Any number of fragments specified by 5′ and 3′positions or by size in nucleotides, as described above, may beexcluded.

The GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polynucleotide fragmentsof the invention comprise from 18 consecutive bases to the full-lengthpolynucleotide sequence encoding the GMG-3, GMG-4, Cluster 1, GMG-6A, orGMG-6B fragments described in Section II of the Preferred Embodiments ofthe Invention. In one aspect of this embodiment, the polynucleotidecomprises at least 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150,155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220,225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290,295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360,365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430,435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500,505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570,575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640,645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710,715, 720, 725, 740, 770, 800, 850, 879, 900, 950, 972, 993, 1000 or 1002consecutive nucleotides of a polynucleotide of the present invention.

In addition to the above preferred nucleic acid sizes, further preferrednucleic acids comprise at least 18 nucleotides, wherein “at least 18” isdefined as any integer between 18 and the integer corresponding to the3′ most nucleotide position of a GMG-3, GMG-4, Cluster 1, GMG-6A, orGMG-6B fragment cDNA herein.

Further included as preferred polynucleotides of the present inventionare nucleic acid fragments at least 18 nucleotides in length, asdescribed above, that are further specified in terms of their 5′ and 3′position. The 5′ and 3′ positions are represented by the positionnumbers set forth in the sequence listing below. For allelic anddegenerate and other variants, position 1 is defined as the 5′ mostnucleotide of the open reading frame (ORF), i.e., the nucleotide “A” ofthe start codon (ATG) with the remaining nucleotides numberedconsecutively. Therefore, every combination of a 5′ and 3′ nucleotideposition that a polynucleotide fragment invention, at least 18contiguous nucleotides in length, could occupy on a GMG-3, GMG-4,Cluster 1, GMG-6A, or GMG-6B fragment polynucleotide of the presentinvention is included in the invention as an individual species. Thepolynucleotide fragments specified by 5′ and 3′ positions can beimmediately envisaged and are therefore not individually listed solelyfor the purpose of not unnecessarily lengthening the specification.

It is noted that the above species of polynucleotide fragments of thepresent invention may alternatively be described by the formula “x toy”; where “x” equals the 5′ most nucleotide position and “y” equals the3′ most nucleotide position of the polynucleotide; and further where “x”equals an integer between 1 and the number of nucleotides of the GMG-3,GMG-4, Cluster 1, GMG-6A, or GMG-6B polynucleotide sequences of thepresent invention minus 18, and where “y” equals an integer between 9and the number of nucleotides of the GMG-3, GMG-4, Cluster 1, GMG-6A, orGMG-6B polynucleotide sequences of the present invention; and where “x”is an integer smaller than “y” by at least 18. Every combination of “x”and “y” positions are included as specific embodiments of the invention.Moreover, the formula “x” to “y” may be modified as “x1-x2” to “y1-y2”,wherein “x1-x2” and “y1-y2” represent positional ranges selected fromany two nucleotide positions of the sequence listing. Alternativeformulas include “x1-x2” to “y” and “x” to “y1-y2”.

These specific embodiments, and other polynucleotide fragmentembodiments described herein may be modified as being “at least”, “equalto”, “equal to or less than”, “less than”, “at least ______ but notgreater than ______” or “from ______ to ______” a specified size orspecified 5′ and/or 3′ positions.

The present invention also provides for the exclusion of any species ofpolynucleotide fragments of the present invention specified by 5′ and 3′positions or polynucleotides specified by size in nucleotides asdescribed above. Any number of fragments specified by 5′ and 3′positions or by size in nucleotides, as described above, may beexcluded.

Variants

In other preferred embodiments, variants of GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B polynucleotides encoding GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B polypeptides are envisioned. Variants ofpolynucleotides, as the term is used herein, are polynucleotides whosesequence differs from a reference polynucleotide. A variant of apolynucleotide may be a naturally occurring variant such as a naturallyoccurring allelic variant, or it may be a variant that is not known tooccur naturally. Such non-naturally occurring variants of thepolynucleotide may be made by mutagenesis techniques, including thoseapplied to polynucleotides, cells or organisms. Generally, differencesare limited so that the nucleotide sequences of the reference and thevariant are closely similar overall and, in many regions, identical.

Polynucleotide variants that comprise a sequence substantially differentfrom those described above but that, due to the degeneracy of thegenetic code, still encode GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bpolypeptides of the present invention are also specifically envisioned.It would also be routine for one skilled in the art to generate thedegenerate variants described above, for instance, to optimize codonexpression for a particular host (e.g., change codons in the human mRNAto those preferred by other mammalian or bacterial host cells).

As stated above, variant polynucleotides may occur naturally, such as anatural allelic variant, or by recombinant methods. By an “allelicvariant” is intended one of several alternate forms of a gene occupyinga given locus on a chromosome of an organism (See, e.g., B. Lewin,(1990) Genes IV, Oxford University Press, New York). Non-naturallyoccurring variants may be produced using art-known mutagenesistechniques. Such nucleic acid variants include those produced bynucleotide substitutions, deletions, or additions. The substitutions,deletions, or additions may involve one or more nucleotides. Alterationsin the coding regions may produce conservative or non-conservative aminoacid substitutions, deletions or additions. Especially preferred amongthese are silent substitutions, additions and deletions, which do notalter the properties and activities of GMG-3, GMG-4, Cluster 1, GMG-6A,or GMG-6B polypeptides of the invention. Also preferred in this regardare conservative substitutions.

Nucleotide changes present in a variant polynucleotide are preferablysilent, which means that they do not alter the amino acids encoded bythe polynucleotide. However, nucleotide changes may also result in aminoacid substitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence.

In cases where the nucleotide substitutions result in one or more aminoacid changes, preferred GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bpolypeptides include those that retain one or more metabolic-relatedactivity as described in Section I of the Preferred Embodiments of theInvention.

By “retain the same activities” is meant that the activity measuredusing the polypeptide encoded by the variant GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B polynucleotide in assays is at least 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, or 100%, and not more than 101%, 102%,103%, 104%, 105%, 110%, 115%, 120% or 125% of the activity measuredusing a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide describedin the Examples Section herein.

By the activity being “increased” is meant that the activity measuredusing the polypeptide encoded by the variant GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B polynucleotide in assays is at least 125%, 130%, 135%,140%, 145%, 150%, 155%, 160%, 170%, 180%, 190%, 200%, 225%, 250%, 275%,300%, 325%, 350%, 375%, 400%, 450%, or 500% of the activity measuredusing a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide describedin the Examples Section herein.

By the activity being “decreased” is meant that the activity measuredusing the polypeptide encoded by the variant GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B polynucleotide in assays is decreased by at least 25%,30%, 35%, 40%, 45%, 50%, 75%, 80%, 90% or 95% of the activity measuredusing a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide describedin the Examples Section herein

Percent Identity

The present invention is further directed to nucleic acid moleculeshaving sequences at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or99% identical to the polynucleotide sequences of SEQ ID NOs: 1, 3, 5, 7,or 9 or fragments thereof that encode a polypeptide havingmetabolic-related activity as described in Section I of the PreferredEmbodiments of the Invention. Of course, due to the degeneracy of thegenetic code, one of ordinary skill in the art will immediatelyrecognize that a large number of the nucleic acid molecules at least50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to thenucleic acid sequences shown in SEQ ID NOs: 1, 3, 5, 7, or 9 orfragments thereof will encode a polypeptide having biological activity.In fact, since degenerate variants of these nucleotide sequences allencode the same polypeptide, this will be clear to the skilled artisaneven without performing the above described comparison assay. It will befurther recognized in the art that, for such nucleic acid molecules thatare not degenerate variants, a reasonable number will also encode apolypeptide having biological activity. This is because the skilledartisan is fully aware of amino acid substitutions that are either lesslikely or not likely to significantly affect protein function (e.g.,replacing one aliphatic amino acid with a second aliphatic amino acid),as further described previously in Section I of the PreferredEmbodiments of the Invention.

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence of the presentinvention, it is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence encoding the GMG-3,GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide. In other words, toobtain a polynucleotide having a nucleotide sequence at least 95%identical to a reference nucleotide sequence, up to 5% of thenucleotides in the reference sequence may be deleted, inserted, orsubstituted with another nucleotide. The query sequence may be an entiresequence or any fragment specified as described herein.

The methods of determining and defining whether any particular nucleicacid molecule or polypeptide is at least 50%, 60%, 70%, 80%, 90%, 95%,96%, 97%, 98% or 99% identical to a nucleotide sequence of the presentinvention can be done by using known computer programs. A preferredmethod for determining the best overall match between a query sequence(a sequence of the present invention) and a subject sequence, alsoreferred to as a global sequence alignment, can be determined using theFASTDB computer program based on the algorithm of Brutlag et al.,((1990) Comput Appl Biosci. July; 6(3):237-45). In a sequence alignmentthe query and subject sequences are both DNA sequences. An RNA sequencecan be compared by first converting U's to T's. The result of saidglobal sequence alignment is in percent identity. Preferred parametersused in a FASTDB alignment of DNA sequences to calculate percentidentity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, JoiningPenalty=30, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5,Gap Size Penalty 0.05, Window Size=500 or the length of the subjectnucleotide sequence, whichever is shorter.

If the subject sequence is shorter than the query sequence because of 5′or 3′ deletions, not because of internal deletions, a manual correctionmust be made to the results. This is because the FASTDB program does notaccount for 5′ and 3′ truncations of the subject sequence whencalculating percent identity. For subject sequences truncated at the 5′or 3′ ends, relative to the query sequence, the percent identity iscorrected by calculating the number of bases of the query sequence thatare 5′ and 3′ of the subject sequence, which are not matched/aligned, asa percent of the total bases of the query sequence. Whether a nucleotideis matched/aligned is determined by results of the FASTDB sequencealignment. This percentage is then subtracted from the percent identity,calculated by the above FASTDB program using the specified parameters,to arrive at a final percent identity score. This corrected score iswhat is used for the purposes of the present invention. Only nucleotidesoutside the 5′ and 3′ nucleotides of the subject sequence, as displayedby the FASTDB alignment, which are not matched/aligned with the querysequence, are calculated for the purposes of manually adjusting thepercent identity score.

For example, a 90-nucleotide subject sequence is aligned to a100-nucleotide query sequence to determine percent identity. Thedeletions occur at the 5′ end of the subject sequence and therefore, theFASTDB alignment does not show a matched/alignment of the first 10nucleotides at 5′ end. The 10 unpaired nucleotides represent 10% of thesequence (number of nucleotides at the 5′ and 3′ ends not matched/totalnumber of nucleotides in the query sequence) so 10% is subtracted fromthe percent identity score calculated by the FASTDB program. If theremaining 90 nucleotides were perfectly matched the final percentidentity would be 90%.

In another example, a 90 nucleotide subject sequence is compared with a100 nucleotide query sequence. This time the deletions are internaldeletions so that there are no nucleotides on the 5′ or 3′ of thesubject sequence which are not matched/aligned with the query. In thiscase the percent identity calculated by FASTDB is not manuallycorrected. Once again, only nucleotides 5′ and 3′ of the subjectsequence which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are made for thepurposes of the present invention.

Fusions

Further included in the present invention are polynucleotides encodingthe polypeptides of the present invention that are fused in frame to thecoding sequences for additional heterologous amino acid sequences. Alsoincluded in the present invention are nucleic acids encodingpolypeptides of the present invention together with additional,non-coding sequences, including for example, but not limited tonon-coding 5′ and 3′ sequences, vector sequence, sequences used forpurification, probing, or priming. For example, heterologous sequencesinclude transcribed, nontranslated sequences that may play a role intranscription, and mRNA processing, for example, ribosome binding andstability of mRNA. The heterologous sequences may alternatively compriseadditional coding sequences that provide additional functionalities.Thus, a nucleotide sequence encoding a polypeptide may be fused to a tagsequence, such as a sequence encoding a peptide that facilitatespurification of the fused polypeptide. In certain preferred embodimentsof this aspect of the invention, the tag amino acid sequence is ahexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Chatsworth, Calif.), among others, many of which arecommercially available. For instance, hexa-histidine provides forconvenient purification of the fusion protein (See, Gentz et al., (1989)Proc Natl Acad Sci USA 86: 821-4). The “HA” tag is another peptideuseful for purification which corresponds to an epitope derived from theinfluenza hemagglutinin protein (See, Wilson et al., (1984) Cell37:767-78). As discussed above, other such fusion proteins includeGMG-3, GMG-4, GMG-6A, or GMG-6B cDNA or Cluster 1 polynucleotide fusedto Fc at the N- or C-terminus.

III. Recombinant Vectors of the Invention

The term “vector” is used herein to designate either a circular or alinear DNA or RNA molecule, that is either double-stranded orsingle-stranded, and that comprises at least one polynucleotide ofinterest that is sought to be transferred in a cell host or in aunicellular or multicellular host organism.

The present invention relates to recombinant vectors comprising any oneof the polynucleotides described herein.

The present invention encompasses a family of recombinant vectors thatcomprise polynucleotides encoding GMG-3, GMG-4, Cluster 1, GMG-6A, orGMG-6B polypeptides of the invention.

In a first preferred embodiment, a recombinant vector of the inventionis used to amplify the inserted polynucleotide in a suitable cell host,this polynucleotide being amplified every time that the recombinantvector replicates. The inserted polynucleotide can be one that encodesGMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides of theinvention.

A second preferred embodiment of the recombinant vectors according tothe invention consists of expression vectors comprising polynucleotidesencoding GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides of theinvention. Within certain embodiments, expression vectors are employedto express a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide ofthe invention, preferably a modified GMG-3, GMG-4, Cluster 1, GMG-6A, orGMG-6B described in the present invention, which can be then purifiedand, for example, be used as a treatment for metabolic-related diseases,or simply to reduce body mass of individuals.

Expression requires that appropriate signals are provided in thevectors, said signals including various regulatory elements, such asenhancers/promoters from both viral and mammalian sources, that driveexpression of the genes of interest in host cells. Dominant drugselection markers for establishing permanent, stable, cell clonesexpressing the products are generally included in the expression vectorsof the invention, as they are elements that link expression of the drugselection markers to expression of the polypeptide.

More particularly, the present invention relates to expression vectorswhich include nucleic acids encoding a GMG-3, GMG-4, Cluster 1, GMG-6A,or GMG-6B polypeptide of the invention, or a modified GMG-3, GMG-4,Cluster 1, GMG-6A, or GMG-6B as described herein, or variants orfragments thereof, under the control of a regulatory sequence selectedamong GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides, oralternatively under the control of an exogenous regulatory sequence.

Consequently, preferred expression vectors of the invention are selectedfrom the group consisting of: (a) a GMG-3, GMG-4, Cluster 1, GMG-6A, orGMG-6B regulatory sequence and driving the expression of a codingpolynucleotide operably linked thereto; and (b) a GMG-3, GMG-4, Cluster1, GMG-6A, or GMG-6B coding sequence of the invention, operably linkedto regulatory sequences allowing its expression in a suitable cell hostand/or host organism.

Some of the elements that can be found in the vectors of the presentinvention are described in further detail in the following sections.

1) General Features of the Expression Vectors of the Invention

A recombinant vector according to the invention comprises, but is notlimited to, a YAC (Yeast Artificial Chromosome), a BAC (BacterialArtificial Chromosome), a phage, a phagemid, a cosmid, a plasmid, oreven a linear DNA molecule which may consist of a chromosomal,non-chromosomal, semi-synthetic or synthetic DNA. Such a recombinantvector can comprise a transcriptional unit comprising an assembly of:

(1) a genetic element or elements having a regulatory role in geneexpression, for example promoters or enhancers. Enhancers are cis-actingelements of DNA, usually from about 10 to 300 bp in length that act onthe promoter to increase the transcription;

(2) a structural or coding sequence which is transcribed into mRNA andeventually translated into a polypeptide, said structural or codingsequence being operably linked to the regulatory elements described in(1); and

(3) appropriate transcription initiation and termination sequences.Structural units intended for use in yeast or eukaryotic expressionsystems preferably include a leader sequence enabling extracellularsecretion of translated protein by a host cell. Alternatively, when arecombinant protein is expressed without a leader or transport sequence,it may include a N-terminal residue. This residue may or may not besubsequently cleaved from the expressed recombinant protein to provide afinal product.

Generally, recombinant expression vectors will include origins ofreplication, selectable markers permitting transformation of the hostcell, and a promoter derived from a highly expressed gene to directtranscription of a downstream structural sequence. The heterologousstructural sequence is assembled in appropriate phase with translationinitiation and termination sequences, and preferably a leader sequencecapable of directing secretion of the translated protein into theperiplasmic space or the extracellular medium. In a specific embodimentwherein the vector is adapted for transfecting and expressing desiredsequences in mammalian host cells, preferred vectors will comprise anorigin of replication in the desired host, a suitable promoter andenhancer, and also any necessary ribosome binding sites, polyadenylationsites, splice donor and acceptor sites, transcriptional terminationsequences, and 5′-flanking non-transcribed sequences. DNA sequencesderived from the SV40 viral genome, for example SV40 origin, earlypromoter, enhancer, splice and polyadenylation sites may be used toprovide the required non-transcribed genetic elements.

2) Regulatory Elements

 Promoters

The suitable promoter regions used in the expression vectors of thepresent invention are chosen taking into account the cell host in whichthe heterologous gene is expressed. The particular promoter employed tocontrol the expression of a nucleic acid sequence of interest is notbelieved to be important, so long as it is capable of directing theexpression of the nucleic acid in the targeted cell. Thus, where a humancell is targeted, it is preferable to position the nucleic acid codingregion adjacent to and under the control of a promoter that is capableof being expressed in a human cell, such as, for example, a human or aviral promoter.

A suitable promoter may be heterologous with respect to the nucleic acidfor which it controls the expression or alternatively can be endogenousto the native polynucleotide containing the coding sequence to beexpressed. Additionally, the promoter is generally heterologous withrespect to the recombinant vector sequences within which the constructpromoter/coding sequence has been inserted.

Promoter regions can be selected from any desired gene using, forexample, CAT (chloramphenicol transferase) vectors and more preferablypKK232-8 and pCM7 vectors. Preferred bacterial promoters are the LacI,LacZ, the T3 or T7 bacteriophage RNA polymerase promoters, the gpt,lambda PR, PL and trp promoters (EP 0036776), the polyhedrin promoter,or the p10 protein promoter from baculovirus (Kit Novagen) (Smith etal., (1983) Mol Cell Biol 3:2156-65; O'Reilly et al., 1992), the lambdaPR promoter or also the trc promoter.

Eukaryotic promoters include CMV immediate early, HSV thymidine kinase,early and late SV40, LTRs from retrovirus, and mouse metallothionein-L.In addition, promoters specific for a particular cell type may bechosen, such as those facilitating expression in adipose tissue, muscletissue, or liver. Selection of a convenient vector and promoter is wellwithin the level of ordinary skill in the art.

The choice of a promoter is well within the ability of a person skilledin the field of genetic engineering. For example, one may refer toSambrook et al. (1989) Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, NY, Vol. 1, 2, 3 (1989), or also to theprocedures described by Fuller et al. (1996) Immunology in CurrentProtocols in Molecular Biology.

 Other Regulatory Elements

Where a cDNA insert is employed, one will typically desire to include apolyadenylation signal to effect proper polyadenylation of the genetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and any suchsequence may be employed such as human growth hormone and SV40polyadenylation signals. Also contemplated as an element of theexpression cassette is a terminator. These elements can serve to enhancemessage levels and to minimize read through from the cassette into othersequences.

Vectors containing the appropriate DNA sequence as described above canbe utilized to transform an appropriate host to allow the expression ofthe desired polypeptide or polynucleotide.

3) Selectable Markers

Such markers would confer an identifiable change to the cell permittingeasy identification of cells containing the expression construct. Theselectable marker genes for selection of transformed host cells arepreferably dihydrofolate reductase or neomycin resistance for eukaryoticcell culture, TRP 1 for S. cerevisiae or tetracycline, rifampicin orampicillin resistance in E. coli, or levan saccharase for mycobacteria,this latter marker being a negative selection marker.

4) Preferred Vectors

 Bacterial Vectors

As a representative but non-limiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and a bacterialorigin of replication derived from commercially available plasmidscomprising genetic elements of pBR322 (ATCC 37017). Such commercialvectors include, for example, pKK223-3 (Pharmacia, Uppsala, Sweden), andpGEM1 (Promega Biotec, Madison, Wis. USA).

Large numbers of other suitable vectors are known to those of skill inthe art, and are commercially available, such as the following bacterialvectors: pQE70, pQE60, pQE-9 (Qiagen), pbs, pD10, phagescript, psiX174,pbluescript SK, pbsks, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene);ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); pWLNEO, pSV2CAT,pOG44, pXT1, pSG (Stratagene); pSVK3, pBPV, pMSG, pSVL (Pharmacia);pQE-30 (QIAexpress).

 Baculovirus Vectors

A suitable vector for the expression of polypeptides of the invention isa baculovirus vector that can be propagated in insect cells and ininsect cell lines. A specific suitable host vector system is thepVL1392/1393 baculovirus transfer vector (Pharmingen) that is used totransfect the SF9 cell line (ATCC No. CRL 1711) which is derived fromSpodoptera frugiperda.

Other suitable vectors for the expression of a GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B polypeptide comprising the C-terminal globular C1qhomology domain in a baculovirus expression system include thosedescribed by Chai et al. (1993; Biotechnol Appl Biochem 18 (Pt3):259-73); Vlasak et al. (1983; Eur J Biochem 135:123-6); and Lenhardet al. (1996; Gene 169:187-90).

 Plasmid Vectors

A suitable vector for the expression of polypeptides of the invention isa plasmid vector that contains an SV40-derived origin of replication andthat can be used for transient transfection of COS cells (ATCC No.CRL1650; No. CRL1651). Plasmid vectors suitable for transienttransfection of COS cells include but are not limited to CDM8(Invitrogen).

 Viral Vectors

In one specific embodiment, the vector is derived from an adenovirus.Preferred adenovirus vectors according to the invention are thosedescribed by Feldman and Steg (1996; Semin Interv Cardiol 1:203-8) orOhno et al. (1994; Science 265:781-4). Another preferred recombinantadenovirus according to this specific embodiment of the presentinvention is the human adenovirus type 2 or 5 (Ad 2 or Ad 5) or anadenovirus of animal origin (French patent application No. FR-93.05954).

Retrovirus vectors and adeno-associated virus vectors are generallyunderstood to be the recombinant gene delivery systems of choice for thetransfer of exogenous polynucleotides in vivo, particularly to mammals,including humans. These vectors provide efficient delivery of genes intocells, and the transferred nucleic acids are stably integrated into thechromosomal DNA of the host.

Particularly preferred retroviruses for the preparation or constructionof retroviral in vitro or in vivo gene delivery vehicles of the presentinvention include retroviruses selected from the group consisting ofMink-Cell Focus Inducing Virus, Murine Sarcoma Virus,Reticuloendotheliosis virus and Rous Sarcoma virus. Particularlypreferred Murine Leukemia Viruses include the 4070A and the 1504Aviruses, Abelson (ATCC No VR-999), Friend (ATCC No VR-245), Gross (ATCCNo VR-590), Rauscher (ATCC No VR-998) and Moloney Murine Leukemia Virus(ATCC No VR-190; PCT Application No WO 94/24298). Particularly preferredRous Sarcoma Viruses include Bryan high titer (ATCC Nos VR-334, VR-657,VR-726, VR-659 and VR-728). Other preferred retroviral vectors are thosedescribed in Roth et al. (1996), PCT Application No WO 93/25234, PCTApplication No WO 94/06920, Roux et al., ((1989) Proc Natl Acad Sci USA86:9079-83), Julan et al., (1992) J. Gen. Virol 3:3251-3255 and Neda etal., ((1991) J Biol Chem 266:14143-6).

Yet another viral vector system that is contemplated by the inventionconsists of the adeno-associated virus (AAV). The adeno-associated virusis a naturally occurring defective virus that requires another virus,such as an adenovirus or a herpes virus, as a helper virus for efficientreplication and a productive life cycle (Muzyczka et al., (1992) CurrTop Microbiol Immunol 158:97-129). It is also one of the few virusesthat may integrate its DNA into non-dividing cells, and exhibits a highfrequency of stable integration (Flotte et al., (1992) Am J Respir CellMol Biol 7:349-56; Samulski et al., (1989) J Virol 63:3822-8; McLaughlinet al., (1989) Am J Hum Genet 59:561-569). One advantageous feature ofAAV derives from its reduced efficacy for transducing primary cellsrelative to transformed cells.

5) Delivery of the Recombinant Vectors

In order to effect expression of the polynucleotides of the invention,these constructs must be delivered into a cell. This delivery may beaccomplished in vitro, as in laboratory procedures for transforming celllines, or in vivo or ex vivo, as in the treatment of certain diseasestates.

One mechanism is viral infection where the expression construct isencapsulated in an infectious viral particle.

Several non-viral methods for the transfer of polynucleotides intocultured mammalian cells are also contemplated by the present invention,and include, without being limited to, calcium phosphate precipitation(Graham et al., (1973) Virology 54:536-9; Chen et al., (1987) Mol CellBiol 7:2745-52), DEAE-dextran (Gopal, (1985) Mol Cell Biol, 5:1188-90),electroporation (Tur-Kaspa et al., (1986) Mol Cell Biol 6:716-8; Potteret al., (1984) Proc Natl Acad Sci USA 81:7161-5.), direct microinjection(Harland et al., (1985) J Cell Biol 101:1094-9), DNA-loaded liposomes(Nicolau et al., (1982) Biochim Biophys Acta 721:185-90; Fraley et al.,(1979) Proc Natl Acad Sci USA 76:3348-52), and receptor-mediatedtransfection (Wu and Wu, (1987) J Biol Chem 262:4429-32; Wu and Wu(1988) Biochemistry 27:887-92). Some of these techniques may besuccessfully adapted for in vivo or ex vivo use.

Once the expression polynucleotide has been delivered into the cell, itmay be stably integrated into the genome of the recipient cell. Thisintegration may be in the cognate location and orientation viahomologous recombination (gene replacement) or it may be integrated in arandom, non-specific location (gene augmentation). In yet furtherembodiments, the nucleic acid may be stably maintained in the cell as aseparate, episomal segment of DNA. Such nucleic acid segments or“episomes” encode sequences sufficient to permit maintenance andreplication independent of or in synchronization with the host cellcycle.

One specific embodiment for a method for delivering a protein or peptideto the interior of a cell of a vertebrate in vivo comprises the step ofintroducing a preparation comprising a physiologically acceptablecarrier and a naked polynucleotide operatively coding for thepolypeptide of interest into the interstitial space of a tissuecomprising the cell, whereby the naked polynucleotide is taken up intothe interior of the cell and has a physiological effect. This isparticularly applicable for transfer in vitro but it may be applied toin vivo as well.

Compositions for use in vitro and in vivo comprising a “naked”polynucleotide are described in PCT application No. WO 90/11092 (VicalInc.) and also in PCT application No. WO 95/11307 (Institut Pasteur,INSERM, Université d'Ottawa) as well as in the articles of Tascon et al.(1996) Nature Medicine 2:888-892 and of Huygen et al. ((1996) Nat Med2:893-8).

In still another embodiment of the invention, the transfer of a nakedpolynucleotide of the invention, including a polynucleotide construct ofthe invention, into cells may be proceeded with a particle bombardment(biolistic), said particles being DNA-coated microprojectilesaccelerated to a high velocity allowing them to pierce cell membranesand enter cells without killing them, such as described by Klein et al.((1990) Curr Genet 17:97-103).

In a further embodiment, the polynucleotide of the invention may beentrapped in a liposome (Ghosh and Bacchawat, (1991) Targeted Diagn Ther4:87-103; Wong et al., (1980) Gene 10:87-94; Nicolau et al., (1987)Methods Enzymol. 149:157-76). These liposomes may further be targeted tocells expressing LSR by incorporating leptin, triglycerides, or otherknown LSR ligands into the liposome membrane.

In a specific embodiment, the invention provides a composition for thein vivo production of a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bglobular head polypeptide described herein. It comprises a nakedpolynucleotide operatively coding for this polypeptide, in solution in aphysiologically acceptable carrier, and suitable for introduction into atissue to cause cells of the tissue to express the said polypeptide.

The amount of vector to be injected to the desired host organism variesaccording to the site of injection. As an indicative dose, it will beinjected between 0.1 and 100 μg of the vector in an animal body,preferably a mammal body, for example a mouse body.

In another embodiment of the vector according to the invention, it maybe introduced in vitro in a host cell, preferably in a host cellpreviously harvested from the animal to be treated and more preferably asomatic cell such as a muscle cell. In a subsequent step, the cell thathas been transformed with the vector coding for the desired GMG-3,GMG-4, Cluster 1, GMG-6A, or GMG-6B globular head polypeptide or thedesired fragment thereof is reintroduced into the animal body in orderto deliver the recombinant protein within the body either locally orsystemically.

IV. Recombinant Cells of the Invention

Another object of the invention consists of host cells recombinant for,i.e., that have been transformed or transfected with one of thepolynucleotides described herein, and more precisely a polynucleotidecomprising a polynucleotide encoding a GMG-3, GMG-4, Cluster 1, GMG-6A,or GMG-6B polypeptide of the invention such as any one of thosedescribed in “Polynucleotides of the Invention”. These polynucleotidescan be present in cells as a result of transient or stable transfection.The invention includes host cells that are transformed (prokaryoticcells) or that are transfected (eukaryotic cells) with a recombinantvector such as any one of those described in “Recombinant Vectors of theInvention”.

Generally, a recombinant host cell of the invention comprises at leastone of the polynucleotides or the recombinant vectors of the inventionthat are described herein.

Preferred host cells used as recipients for the recombinant vectors ofthe invention are the following:

a) Prokaryotic host cells: Escherichia coli strains (I.E. DH5-α strain),Bacillus subtilis, Salmonella typhimurium, and strains from species likePseudomonas, Streptomyces and Staphylococcus, and

b) Eukaryotic host cells: HeLa cells (ATCC No. CCL2; No. CCL2.1; No.CCL2.2), Cv 1 cells (ATCC No. CCL70), COS cells (ATCC No. CRL1650; No.CRL1651), Sf-9 cells (ATCC No. CRL1711), C127 cells (ATCC No. CRL-1804),3T3 (ATCC No. CRL-6361), CHO (ATCC No. CCL-61), human kidney 293 (ATCCNo. 45504; No. CRL-1573), BHK (ECACC No. 84100501; No. 84111301), PLCcells, HepG2, and Hep3B.

The constructs in the host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.

Following transformation of a suitable host and growth of the host to anappropriate cell density, the selected promoter is induced byappropriate means, such as temperature shift or chemical induction, andcells are cultivated for an additional period.

In a preferred embodiment, recombinant protein expressed by cells thathave been stably or transiently transfected with a recombinant vectorsuch as any one of those described in “recombinant Vectors of theInvention” is recovered from culture supernatant.

Alternatively, cells may be harvested (typically by centrifugation),disrupted by physical or chemical means, and the resulting crude extractretained for further purification.

Microbial cells employed in the expression of proteins can be disruptedby any convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents. Such methods arewell known by the skilled artisan.

Further, according to the invention, these recombinant cells can becreated in vitro or in vivo in an animal, preferably a mammal, mostpreferably selected from the group consisting of mice, rats, dogs, pigs,sheep, cattle, and primates, not to include humans. Recombinant cellscreated in vitro can also be later surgically implanted in an animal,for example. Methods to create recombinant cells in vivo in animals arewell-known in the art.

The present invention also encompasses primary, secondary, andimmortalized homologously recombinant host cells of vertebrate origin,preferably mammalian origin and particularly human origin, that havebeen engineered to: a) insert exogenous (heterologous) polynucleotidesinto the endogenous chromosomal DNA of a targeted gene, b) deleteendogenous chromosomal DNA, and/or c) replace endogenous chromosomal DNAwith exogenous polynucleotides. Insertions, deletions, and/orreplacements of polynucleotide sequences may be to the coding sequencesof the targeted gene and/or to regulatory regions, such as promoter andenhancer sequences, operably associated with the targeted gene.

The present invention further relates to a method of making ahomologously recombinant host cell in vitro or in vivo, wherein theexpression of a targeted gene not normally expressed in the cell isaltered. Preferably the alteration causes expression of the targetedgene under normal growth conditions or under conditions suitable forproducing the polypeptide encoded by the targeted gene. The methodcomprises the steps of: (a) transfecting the cell in vitro or in vivowith a polynucleotide construct, the polynucleotide constructcomprising; (i) a targeting sequence; (ii) a regulatory sequence and/ora coding sequence; and (iii) an unpaired splice donor site, ifnecessary, thereby producing a transfected cell; and (b) maintaining thetransfected cell in vitro or in vivo under conditions appropriate forhomologous recombination.

The present invention further relates to a method of altering theexpression of a targeted gene in a cell in vitro or in vivo wherein thegene is not normally expressed in the cell, comprising the steps of: (a)transfecting the cell in vitro or in vivo with a polynucleotideconstruct, the polynucleotide construct comprising: (i) a targetingsequence; (ii) a regulatory sequence and/or a coding sequence; and (iii)an unpaired splice donor site, if necessary, thereby producing atransfected cell; and (b) maintaining the transfected cell in vitro orin vivo under conditions appropriate for homologous recombination,thereby producing a homologously recombinant cell; and (c) maintainingthe homologously recombinant cell in vitro or in vivo under conditionsappropriate for expression of the gene.

The present invention further relates to a method of making apolypeptide of the present invention by altering the expression of atargeted endogenous gene in a cell in vitro or in vivo wherein the geneis not normally expressed in the cell, comprising the steps of: a)transfecting the cell in vitro with a polynucleotide construct, thepolynucleotide construct comprising: (i) a targeting sequence; (ii) aregulatory sequence and/or a coding sequence; and (iii) an unpairedsplice donor site, if necessary, thereby producing a transfected cell;(b) maintaining the transfected cell in vitro or in vivo underconditions appropriate for homologous recombination, thereby producing ahomologously recombinant cell; and c) maintaining the homologouslyrecombinant cell in vitro or in vivo under conditions appropriate forexpression of the gene thereby making the polypeptide.

The present invention further relates to a polynucleotide construct thatalters the expression of a targeted gene in a cell type in which thegene is not normally expressed. This occurs when a polynucleotideconstruct is inserted into the chromosomal DNA of the target cell,wherein the polynucleotide construct comprises: a) a targeting sequence;b) a regulatory sequence and/or coding sequence; and c) an unpairedsplice-donor site, if necessary. Further included are polynucleotideconstructs, as described above, wherein the construct further comprisesa polynucleotide that encodes a polypeptide and is in-frame with thetargeted endogenous gene after homologous recombination with chromosomalDNA.

The compositions may be produced, and methods performed, by techniquesknown in the art, such as those described in U.S. Pat. Nos. 6,054,288;6,048,729; 6,048,724; 6,048,524; 5,994,127; 5,968,502; 5,965,125;5,869,239; 5,817,789; 5,783,385; 5,733,761; 5,641,670; 5,580,734;International Publication Nos: WO96/29411, WO 94/12650; and scientificarticles described by Koller et al., (1994) Annu Rev Immunol 10:705-730;the disclosures of each of which are incorporated by reference in theirentireties).

The expression of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B inmammalian, and typically human, cells may be rendered defective, oralternatively it may be enhanced, with the insertion of a GMG-3, GMG-4,Cluster 1, GMG-6A, or GMG-6B genomic or cDNA sequence with thereplacement of the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B genecounterpart in the genome of an animal cell by a GMG-3, GMG-4, Cluster1, GMG-6A, or GMG-6B polynucleotide according to the invention. Thesegenetic alterations may be generated by homologous recombination eventsusing specific DNA constructs that have been previously described.

One kind of host cell that may be used are mammalian zygotes, such asmurine zygotes. For example, murine zygotes may undergo microinjectionwith a purified DNA molecule of interest, for example a purified DNAmolecule that has previously been adjusted to a concentration range from1 ng/ml—for BAC inserts—3 ng/μl—for P1 bacteriophage inserts—in 10 mMTris-HCl, pH 7.4, 250 μM EDTA containing 100 mM NaCl, 30 μM spermine,and 70 μM spermidine. When the DNA to be microinjected has a large size,polyamines and high salt concentrations can be used in order to avoidmechanical breakage of this DNA, as described by Schedl et al ((1993)Nature 362:258-61).

Any one of the polynucleotides of the invention, including the DNAconstructs described herein, may be introduced in an embryonic stem (ES)cell line, preferably a mouse ES cell line. ES cell lines are derivedfrom pluripoient, uncommitted cells of the inner cell mass ofpre-implantation blastocysts. Preferred ES cell lines are the following:ES-E14TG2a (ATCC No. CRL-1821), ES-D3 (ATCC No. CRL1934 and No.CRL-11632), YS001 (ATCC No. CRL-11776), 36.5 (ATCC No. CRL-11116). Tomaintain ES cells in an uncommitted state, they are cultured in thepresence of growth inhibited feeder cells which provide the appropriatesignals to preserve this embryonic phenotype and serve as a matrix forES cell adherence. Preferred feeder cells are primary embryonicfibroblasts that are established from tissue of day 13-day 14 embryos ofvirtually any mouse strain, that are maintained in culture, such asdescribed by Abbondanzo et al. (1993; Methods Enzymol 225:803-23) andare inhibited in growth by irradiation, such as described by Robertson((1987) Embryo-derived stem cell lines. In: E. J. Robertson Ed.Teratocarcinomas and embrionic stem cells: a practical approach. IRLPress, Oxford), or by the presence of an inhibitory concentration ofLIF, such as described by Pease and Williams (1990; Exp Cell Res190:209-11).

The constructs in the host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.

Following transformation of a suitable host and growth of the host to anappropriate cell density, the selected promoter is induced byappropriate means, such as temperature shift or chemical induction, andcells are cultivated for an additional period. Cells are typicallyharvested by centrifugation, disrupted by physical or chemical means,and the resulting crude extract retained for further purification.Microbial cells employed in the expression of proteins can be disruptedby any convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents. Such methods arewell known by the skilled artisan.

V. DNA Construct that Enables Directing Temporal and Spatial GMG-3.GMG-4, Cluster 1, GMG-6A, or GMG-6B Gene Expression in TransgenicAnimals

DNA Constructs Allowing Homologous Recombination: Replacement Vectors

A preferred DNA construct will comprise, from 5′-end to 3′-end: (a) afirst nucleotide sequence that is comprised in the GMG-3, GMG-4, Cluster1, GMG-6A, or GMG-6B genomic sequence; (b) a nucleotide sequencecomprising a positive selection marker, such as the marker for neomycineresistance (neo); and (c) a second nucleotide sequence that is comprisedin the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B genomic sequence, andis located on the genome downstream the first GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B nucleotide sequence (a).

In a preferred embodiment, this DNA construct also comprises a negativeselection marker located upstream the nucleotide sequence (a) ordownstream the nucleotide sequence (c). Preferably, the negativeselection marker comprises the thymidine kinase (tk) gene (Thomas etal., 1986), the hygromycine beta gene (Te Riele et al., 1990), the hprtgene (Van der Lugt et al., 1991; Reid et al., 1990) or the Diphteriatoxin A fragment (Dt-A) gene (Nada et al., 1993; Yagi et al. 1990),which disclosures are hereby incorporated by reference in theirentireties. Preferably, the positive selection marker is located withina GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B exon sequence so as tointerrupt the sequence encoding a GMG-3, GMG-4, Cluster 1, GMG-6A, orGMG-6B protein. These replacement vectors are described, for example, byThomas et al. (1986; 1987), Mansour et al. (1988) and Koller et al.(1992).

The first and second nucleotide sequences (a) and (c) may beindifferently located within a GMG-3, GMG-4, Cluster 1, GMG-6A, orGMG-6B regulatory sequence, an intronic sequence, an exon sequence or asequence containing both regulatory and/or intronic and/or exonsequences. The size of the nucleotide sequences (a) and (c) ranges from1 to 50 kb, preferably from 1 to 10 kb, more preferably from 2 to 6 kband most preferably from 2 to 4 kb. Preferably the nucleotide sequences(a) and (c) comprise SEQ ID NO: 11, 12, or 13.

DNA Constructs Allowing Homologous Recombination: Cre-LoxP System

These new DNA constructs make use of the site specific recombinationsystem of the P1 phage. The P1 phage possesses a recombinase called Crethat interacts specifically with a 34 base pairs loxP site. The loxPsite is composed of two palindromic sequences of 13 bp separated by a 8bp conserved sequence (Hoess et al., 1986), which disclosure is herebyincorporated by reference in its entirety. The recombination by the Creenzyme between two loxP sites having an identical orientation leads tothe deletion of the DNA fragment.

The Cre-loxP system used in combination with a homologous recombinationtechnique has been first described by Gu et al. (1993, 1994), whichdisclosures are hereby incorporated by reference in their entireties.Briefly, a nucleotide sequence of interest to be inserted in a targetedlocation of the genome harbors at least two loxP sites in the sameorientation and located at the respective ends of a nucleotide sequenceto be excised from the recombinant genome. The excision event requiresthe presence of the recombinase (Cre) enzyme within the nucleus of therecombinant cell host. The recombinase enzyme may be brought at thedesired time either by (a) incubating the recombinant cell hosts in aculture medium containing this enzyme, by injecting the Cre enzymedirectly into the desired cell, such as described by Araki et al (1995),which disclosure is hereby incorporated by reference in its entirety, orby lipofection of the enzyme into the cells, such as described byBaubonis et al (1993), which disclosure is hereby incorporated byreference in its entirety; (b) transfecting the cell host with a vectorcomprising the Cre coding sequence operably linked to a promoterfunctional in the recombinant cell host, which promoter being optionallyinducible, said vector being introduced in the recombinant cell host,such as described by Gu et al. (1993) and Sauer et al (1988), whichdisclosures are hereby incorporated by reference in their entireties;(c) introducing in the genome of the cell host a polynucleotidecomprising the Cre coding sequence operably linked to a promoterfunctional in the recombinant cell host, which promoter is optionallyinducible, and said polynucleotide being inserted in the genome of thecell host either by a random insertion event or an homologousrecombination event, such as described by Gu et at. (1994).

In a specific embodiment, the vector containing the sequence to beinserted in the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B gene byhomologous recombination is constructed in such a way that selectablemarkers are flanked by loxP sites of the same orientation, it ispossible, by treatment by the Cre enzyme, to eliminate the selectablemarkers while leaving the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bsequences of interest that have been inserted by an homologousrecombination event. Again, two selectable markers are needed: apositive selection marker to select for the recombination event and anegative selection marker to select for the homologous recombinationevent. Vectors and methods using the Cre-loxP system are described byZou et al. (1994), which disclosure is hereby incorporated by referencein its entirety.

Thus, a second preferred DNA construct of the invention comprises, from5′-end to 3′-end: (a) a first nucleotide sequence that is comprised inthe GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B genomic sequence; (b) anucleotide sequence comprising a polynucleotide encoding a positiveselection marker, said nucleotide sequence comprising additionally twosequences defining a site recognized by a recombinase, such as a loxPsite, the two sites being placed in the same orientation; and (c) asecond nucleotide sequence that is comprised in the GMG-3, GMG-4,Cluster 1, GMG-6A, or GMG-6B genomic sequence, and is located on thegenome downstream of the first GMG-3, GMG-4, Cluster 1, GMG-6A, orGMG-6B nucleotide sequence (a). Preferably the nucleotide sequences (a)and (c) comprise SEQ ID NO: 11, 12, or 13.

The sequences defining a site recognized by a recombinase, such as aloxP site, are preferably located within the nucleotide sequence (b) atsuitable locations bordering the nucleotide sequence for which theconditional excision is sought. In one specific embodiment, two loxPsites are located at each side of the positive selection markersequence, in order to allow its excision at a desired time after theoccurrence of the homologous recombination event.

In a preferred embodiment of a method using the third DNA constructdescribed above, the excision of the polynucleotide fragment bordered bythe two sites recognized by a recombinase, preferably two loxP sites, isperformed at a desired time, due to the presence within the genome ofthe recombinant host cell of a sequence encoding the Cre enzyme operablylinked to a promoter sequence, preferably an inducible promoter, morepreferably a tissue-specific promoter sequence and most preferably apromoter sequence which is both inducible and tissue-specific, such asdescribed by Gu et al (1994).

The presence of the Cre enzyme within the genome of the recombinant cellhost may result from the breeding of two transgenic animals, the firsttransgenic animal bearing the GMG-3-, GMG-4-, Cluster 1-, GMG-6A-, orGMG-6B-derived sequence of interest containing the loxP sites asdescribed above and the second transgenic animal bearing the Cre codingsequence operably linked to a suitable promoter sequence, such asdescribed by Gu et al. (1994).

Spatio-temporal control of the Cre enzyme expression may also beachieved with an adenovirus based vector that contains the Cre gene thusallowing infection of cells, or in vivo infection of organs, fordelivery of the Cre enzyme, such as described by Anton and Graham (1995)and Kanegae et al (1995), which disclosures are hereby incorporated byreference in their entireties.

The DNA constructs described above may be used to introduce a desirednucleotide sequence of the invention, preferably a GMG-3, GMG-4, Cluster1, GMG-6A, or GMG-6B genomic sequence or a GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B cDNA sequence, and most preferably an altered copy ofa GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B genomic or cDNA sequence,within a predetermined location of the targeted genome, leading eitherto the generation of an altered copy of a targeted gene (knock-outhomologous recombination) or to the replacement of a copy of thetargeted gene by another copy sufficiently homologous to allow anhomologous recombination event to occur (knock-in homologousrecombination).

VI. Transgenic Animals

The present invention also provides methods and compositions for thegeneration of non-human animals and plants that express the recombinantGMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides, of the presentinvention. The animals or plants can be transgenic, i.e. each of theircells contains a gene encoding a GMG-3, GMG-4, Cluster 1, GMG-6A, orGMG-6B polypeptide, or, alternatively, a polynucleotide encoding aGMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide can be introducedinto somatic cells of the animal or plant, e.g. into mammary secretoryepithelial cells of a mammal. In preferred embodiments, the non-humananimal is a mammal such as a cow, sheep, goat, pig, or rabbit.

Methods of making transgenic animals such as mammals are well known tothose of skill in the art, and any such method can be used in thepresent invention. Briefly, transgenic mammals can be produced, e.g., bytransfecting a pluripotential stem cell such as an ES cell with apolynucleotide encoding a polypeptide of interest. Successfullytransformed ES cells can then be introduced into an early stage embryothat is then implanted into the uterus of a mammal of the same species.In certain cases, the transformed (“transgenic”) cells will comprisepart of the germ line of the resulting animal, and adult animalscomprising the transgenic cells in the germ line can then be mated toother animals, thereby eventually producing a population of transgenicanimals that have the transgene in each of their cells, and which canstably transmit the transgene to each of their offspring. Other methodsof introducing the polynucleotide can be used, for example introducingthe polynucleotide encoding the polypeptide of interest into afertilized egg or early stage embryo via microinjection. Alternatively,the transgene may be introduced into an animal by infection of zygoteswith a retrovirus containing the transgene (Jaenisch, R. (1976) Proc.Natl. Acad. Sci. USA 73, 1260-1264). Methods of making transgenicmammals are described, e.g., in Wall et al. (1992) J Cell Biochem 199249:113-20; Hogan, et al. (1986) in Manipulating the mouse embryo. ALaboratory Manual. Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.; in WO 91/08216, or in U.S. Pat. No. 4,736,866.

In a preferred method, the polynucleotides are microinjected into thefertilized oocyte. Typically, fertilized oocytes are microinjected usingstandard techniques, and then cultured in vitrountil a “pre-implantationembryo” is obtained. Such pre-implantation embryos preferably containapproximately 16 to 150 cells. Methods for culturing fertilized oocytesto the pre-implantation stage are described, e.g., by Gordon et al.((1984) Methods in Enzymology, 101, 414); Hogan et al. ((1986) inManipulating the mouse embryo. A Laboratory Manual. Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.) (for the mouse embryo);Hammer et al. ((1985) Nature, 315, 680) (for rabbit and porcineembryos); Gandolfi et al. ((1987) J. Reprod. Fert. 81, 23-28); Rexroadet al. ((1988) J. Anim. Sci. 66, 947-953) (for ovine embryos); andEyestone et al. ((1989) J. Reprod. Fert. 85, 715-720); Camous et al.((1984) J. Reprod. Pert. 72, 779-785); and Heyman et al. ((1987)Theriogenology 27, 5968) (for bovine embryos); the disclosures of eachof which are incorporated herein in their entireties. Pre-implantationembryos are then transferred to an appropriate female by standardmethods to permit the birth of a transgenic or chimeric animal,depending upon the stage of development when the transgene isintroduced.

As the frequency of transgene incorporation is often low, the detectionof transgene integration in pre-implantation embryos is often desirableusing any of the herein-described methods. Any of a number of methodscan be used to detect the presence of a transgene in a pre-implantationembryo. For example, one or more cells may be removed from thepre-implantation embryo, and the presence or absence of the transgene inthe removed cell or cells can be detected using any standard method e.g.PCR. Alternatively, the presence of a transgene can be detected in uteroor post partum using standard methods.

In a particularly preferred embodiment of the present invention,transgenic mammals are generated that secrete recombinant GMG-3, GMG-4,Cluster 1, GMG-6A, or GMG-6B polypeptides in their milk. As the mammarygland is a highly efficient protein-producing organ, such methods can beused to produce protein concentrations in the gram per liter range, andoften significantly more. Preferably, expression in the mammary gland isaccomplished by operably linking the polynucleotide encoding the GMG-3,GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide to a mammary glandspecific promoter and, optionally, other regulatory elements. Suitablepromoters and other elements include, but are not limited to, thosederived from mammalian short and long WAP, alpha, beta, and kappa,casein, alpha and beta lactoglobulin, beta-CN 5′ genes, as well as thethe mouse mammary tumor virus (MMTV) promoter. Such promoters and otherelements may be derived from any mammal, including, but not limited to,cows, goats, sheep, pigs, mice, rabbits, and guinea pigs. Promoter andother regulatory sequences, vectors, and other relevant teachings areprovided, e.g., by Clark (1998) J Mammary Gland Biol Neoplasia 3:337-50;Jost et al. (1999) Nat Biotechnol 17:160-4; U.S. Pat. Nos. 5,994,616;6,140,552; 6,013,857; Sohn et al. (1999) DNA Cell Biol. 18:845-52; Kimet al. (1999) J Biochem (Japan) 126:320-5; Soulier et al. (1999) Euro JBiochem 260:533-9; Zhang et al. (1997) Chin J Biotech 13:271-6; Rijnkelset al. (1998) Transgen Res 7:5-14; Korhonen et al. (1997) Euro J Biochem245:482-9; Uusi-Oukari et al. (1997) Transgen Res 6:75-84; Hitchin etal. (1996) Prot Expr Purif 7:247-52; Platenburg et al. (1994) TransgenRes 3:99-108; Heng-Cherl et al. (1993) Animal Biotech 4:89-107; andChrista et al. (2000) Euro J Biochem 267:1665-71; the entire disclosuresof each of which is herein incorporated by reference.

In another embodiment, the polypeptides of the invention can be producedin milk by introducing polynucleotides encoding the polypeptides intosomatic cells of the mammary gland in vivo, e.g. mammary secretingepithelial cells. For example, plasmid DNA can be infused through thenipple canal, e.g. in association with DEAE-dextran (see, e.g., Hens etal. (2000) Biochim. Biophys. Acta 1523:161-171), in association with aligand that can lead to receptor-mediated endocytosis of the construct(see, e.g., Sobolev et al. (1998) 273:7928-33), or in a viral vectorsuch as a retroviral vector, e.g. the Gibbon ape leukemia virus (see,e.g., Archer et al. (1994) PNAS 91:6840-6844). In any of theseembodiments, the polynucleotide may be operably linked to a mammarygland specific promoter, as described above, or, alternatively, anystrongly expressing promoter such as CMV or MoMLV LTR.

The suitability of any vector, promoter, regulatory element, etc. foruse in the present invention can be assessed beforehand by transfectingcells such as mammary epithelial cells, e.g. MacT cells (bovine mammaryepithelial cells) or GME cells (goat mammary epithelial cells), in vitroand assessing the efficiency of transfection and expression of thetransgene in the cells.

For in vivo administration, the polynucleotides can be administered inany suitable formulation, at any of a range of concentrations (e.g.1-500 μg/ml, preferably 50-100 μg/ml), at any volume (e.g. 1-100 ml,preferably 1 to 20 ml), and can be administered any number of times(e.g. 1, 2, 3, 5, or 10 times), at any frequency (e.g. every 1, 2, 3, 5,10, or any number of days). Suitable concentrations, frequencies, modesof administration, etc. will depend upon the particular polynucleotide,vector, animal, etc., and can readily be determined by one of skill inthe art.

In a preferred embodiment, a retroviral vector such as as Gibbon apeleukemia viral vector is used, as described in Archer et al. ((1994)PNAS 91:6840-6844). As retroviral infection typically requires celldivision, cell division in the mammary glands can be stimulated inconjunction with the administration of the vector, e.g. using a factorsuch as estrodiol benzoate, progesterone, reserpine, or dexamethasone.Further, retroviral and other methods of infection can be facilitatedusing accessory compounds such as polybrene.

In any of the herein-described methods for obtaining GMG-3, GMG-4,Cluster 1, GMG-6A, or GMG-6B polypeptides from milk, the quantity ofmilk obtained, and thus the quantity of GMG-3, GMG-4, Cluster 1, GMG-6A,or GMG-6B polypeptides produced, can be enhanced using any standardmethod of lactation induction, e.g. using hexestrol, estrogen, and/orprogesterone.

The polynucleotides used in such embodiments can either encode afull-length GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide or aGMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B fragment. Typically, theencoded polypeptide will include a signal sequence to ensure thesecretion of the protein into the milk. Where a full length GMG-3,GMG-4, Cluster 1, GMG-6A, or GMG-6B sequence is used, the full lengthprotein can, e.g., be isolated from milk and cleaved in vitro using asuitable protease. Alternatively, a second, protease-encodingpolynucleotide can be introduced into the animal or into the mammarygland cells, whereby expression of the protease results in the cleavageof the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide in vivo,thereby allowing the direct isolation of GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B fragments from milk.

VII Pharmaceutical or Physiologically Acceptable Compositions of theInvention

The GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides of theinvention can be administered to non-human animals and/or humans, aloneor in pharmaceutical or physiologically acceptable compositions wherethey are mixed with suitable carriers or excipient(s). Thepharmaceutical or physiologically acceptable composition is thenprovided at a therapeutically effective dose. A therapeuticallyeffective dose refers to that amount of a GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG 6B polypeptide sufficient to result in prevention oramelioration of symptoms or physiological status of metabolic-relateddiseases or disorders as determined by the methods described herein. Atherapeutically effective dose can also refer to the amount of a GMG-3,GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide necessary for areduction in weight or a prevention of an increase in weight orprevention of an increase in the rate of weight gain in persons desiringthis affect for cosmetic reasons. A therapeutically effective dosage ofa GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide of theinvention is that dosage that is adequate to promote weight loss orweight gain with continued periodic use or administration. Techniquesfor formulation and administration of GMG-3, GMG-4, Cluster 1, GMG-6A,or GMG-6B polypeptides may be found in “Remington's PharmaceuticalSciences,” Mack Publishing Co., Easton, Pa., latest edition.

Other diseases or disorders that GMG-3, GMG-4, Cluster 1, GMG-6A, orGMG-6B polypeptides of the invention could be used to treat or preventinclude, but are not limited to, obesity and obesity-related diseasesand disorders such as obesity, impaired glucose tolerance, insulinresistance, atherosclerosis, atheromatous disease, heart disease,hypertension, stroke, Syndrome X, Non-Insulin Dependent DiabetesMellitus (NIDDM, or Type II diabetes) and Insulin Dependent DiabetesMellitus (IDDM or Type I diabetes). Diabetes-related complications to betreated by the methods of the invention include microangiopathiclesions, ocular lesions, retinopathy, neuropathy, renal lesions. Heartdisease includes, but is not limited to, cardiac insufficiency, coronaryinsufficiency, and high blood pressure. Other obesity-related disordersto be treated by compounds of the invention include hyperlipidemia andhyperuricemia. Yet other obesity-related diseases or disorders of theinvention include cachexia, wasting, AIDS-related weight loss,cancer-related weight loss, anorexia, and bulimia. The GMG-3, GMG-4,Cluster 1, GMG-6A, or GMG-6B polypeptides may also be used to enhancephysical performance during work or exercise or enhance a feeling ofgeneral well-being. Physical performance activities include walking,running, jumping, lifting and/or climbing.

The GMG-3, GMG-4, Cluster1, GMG-6A, or GMG-6B polypeptides orantagonists thereof may also be used to treat dyslexia,attention-deficit disorder (ADD), attention-deficit/hyperactivitydisorder (ADHD), and psychiatric disorders such as schizophrenia bymodulating fatty acid metabolism, more specifically, the production ofcertain long-chain polyunsaturated fatty acids.

It is expressly considered that the GMG-3, GMG-4, Cluster 1, GMG-6A, orGMG-6B polypeptides of the invention may be provided alone or incombination with other pharmaceutically or physiologically acceptablecompounds. Other compounds useful for the treatment of obesity and otherdiseases and disorders are currently well-known in the art.

In a preferred embodiment, the GMG-3, GMG-4, Cluster 1, GMG-6A, orGMG-6B polypeptides are useful for, and used in, the treatment ofinsulin resistance and diabetes using methods described herein and knownin the art. More particularly, a preferred embodiments relates toprocess for the therapeutic modification and regulation of glucosemetabolism in an animal or human subject, which comprises administeringto a subject in need of treatment (alternatively on a timed daily basis)GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide (orpolynucleotide encoding said polypeptide) in dosage amount and for aperiod sufficient to reduce plasma glucose levels in said animal orhuman subject.

Further preferred embodiments relate to methods for the prophylaxis ortreatment of diabetes comprising administering to a subject in need oftreatment (alternatively on a timed daily basis) a GMG-3, GMG-4, Cluster1, GMG-6A, or GMG-6B polypeptide (or polynucleotide encoding saidpolypeptide) in dosage amount and for a period sufficient to reduceplasma glucose levels in said animal or human subject.

Routes of Administration.

Suitable routes of administration include oral, nasal, rectal,transmucosal, or intestinal administration, parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, intrapulmonary (inhaled) or intraocularinjections using methods known in the art. A particularly useful methodof administering compounds for promoting weight loss involves surgicalimplantation, for example into the abdominal cavity of the recipient, ofa device for delivering GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bpolypeptides over an extended period of time. Other particularlypreferred routes of administration are aerosol and depot formulation.Sustained release formulations, particularly depot, of the inventedmedicaments are expressly contemplated.

Composition/Formulation

Pharmaceutical or physiologically acceptable compositions andmedicaments for use in accordance with the present invention may beformulated in a conventional manner using one or more physiologicallyacceptable carriers comprising excipients and auxiliaries. Properformulation is dependent upon the route of administration chosen.

Certain of the medicaments described herein will include apharmaceutically or physiologically acceptable acceptable carrier and atleast one polypeptide that is a GMG-3, GMG-4, Cluster 1, GMG-6A, orGMG-6B polypeptide of the invention. For injection, the agents of theinvention may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks's solution, Ringer'ssolution, or physiological saline buffer such as a phosphate orbicarbonate buffer. For transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

Pharmaceutical or physiologically acceptable preparations that can betaken orally include push-fit capsules made of gelatin, as well as soft,sealed capsules made of gelatin and a plasticizer, such as glycerol orsorbitol. The push-fit capsules can contain the active ingredients inadmixture with fillers such as lactose, binders such as starches, and/orlubricants such as talc or magnesium stearate and, optionally,stabilizers. In soft capsules, the active compounds may be dissolved orsuspended in suitable liquids, such as fatty oils, liquid paraffin, orliquid polyethylene glycols. In addition, stabilizers may be added. Allformulations for oral administration should be in dosages suitable forsuch administration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable gaseous propellant, e.g., carbon dioxide. In the case of apressurized aerosol the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin, for use in an inhaler or insufflator, may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g. by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical or physiologically acceptable formulations for parenteraladministration include aqueous solutions of the active compounds inwater-soluble form. Aqueous suspensions may contain substances thatincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents that increase the solubility ofthe compounds to allow for the preparation of highly concentratedsolutions.

Alternatively, the active ingredient may be in powder or lyophilizedform for constitution with a suitable vehicle, such as sterilepyrogen-free water, before use.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

Additionally, the compounds may be delivered using a sustained-releasesystem, such as semipermeable matrices of solid hydrophobic polymerscontaining the therapeutic agent. Various sustained release materialshave been established and are well known by those skilled in the art.Sustained-release capsules may, depending on their chemical nature,release the compounds for a few weeks up to over 100 days.

Depending on the chemical nature and the biological stability of thetherapeutic reagent, additional strategies for protein stabilization maybe employed.

The pharmaceutical or physiologically acceptable compositions also maycomprise suitable solid or gel phase carriers or excipients. Examples ofsuch carriers or excipients include but are not limited to calciumcarbonate, calcium phosphate, various sugars, starches, cellulosederivatives, gelatin, and polymers such as polyethylene glycols.

Effective Dosage.

Pharmaceutical or physiologically acceptable compositions suitable foruse in the present invention include compositions wherein the activeingredients are contained in an effective amount to achieve theirintended purpose. More specifically, a therapeutically effective amountmeans an amount effective to prevent development of or to alleviate theexisting symptoms of the subject being treated. Determination of theeffective amounts is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein.

For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. For example, a dose can be formulated in animal modelsto achieve a circulating concentration range that includes orencompasses a concentration point or range shown to increase leptin orlipoprotein uptake or binding in an in vitro system. Such informationcan be used to more accurately determine useful doses in humans.

A therapeutically effective dose refers to that amount of the compoundthat results in amelioration of symptoms in a patient. Toxicity andtherapeutic efficacy of such compounds can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., for determining the LD50, (the dose lethal to 50% of the testpopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio between LD5Oand ED5O. Compounds that exhibit high therapeutic indices are preferred.

The data obtained from these cell culture assays and animal studies canbe used in formulating a range of dosage for use in humans. The dosageof such compounds lies preferably within a range of circulatingconcentrations that include the ED50, with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. The exactformulation, route of administration and dosage can be chosen by theindividual physician in view of the patient's condition. (See, e.g.,Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch.1).

Dosage amount and interval may be adjusted individually to provideplasma levels of the active compound which are sufficient to maintain orprevent weight loss or gain, depending on the particular situation.Dosages necessary to achieve these effects will depend on individualcharacteristics and route of administration.

Dosage intervals can also be determined using the value for the minimumeffective concentration. Compounds should be administered using aregimen that maintains plasma levels above the minimum effectiveconcentration for 10-90% of the time, preferably between 30-90%; andmost preferably between 50-90%. In cases of local administration orselective uptake, the effective local concentration of the drug may notbe related to plasma concentration.

The amount of composition administered will, of course, be dependent onthe subject being treated, on the subject's weight, the severity of theaffliction, the manner of administration and the judgment of theprescribing physician.

A preferred dosage range for the amount of a GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B polypeptide of the invention, which can beadministered on a daily or regular basis to achieve desired results,including a reduction in levels of circulating plasma triglyceride-richlipoproteins, range from 0.05-1.0 mg/kg body mass. A more preferreddosage range is from 0.1-5 mg/kg. A more preferred dose is 0.25-2.5mg/kg. Of course, these daily dosages can be delivered or administeredin small amounts periodically during the course of a day. It is notedthat these dosage ranges are only preferred ranges and are not meant tobe limiting to the invention.

VIII. Methods of Treatment

The invention is drawn inter alia to methods of preventing or treatingmetabolic-related diseases and disorders comprising providing anindividual in need of such treatment with a GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B polypeptide of the invention. Preferably, the GMG-3,GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide has metabolic-relatedactivity either in vitro or in vivo. Preferably the GMG-3, GMG-4,Cluster 1, GMG-6A, or GMG-6B polypeptide is provided to the individualin a pharmaceutical composition that is preferably taken orally.Preferably the individual is a mammal, and most preferably a human. Inpreferred embodiments, the metabolic-related disease or disorder isselected from the group consisting of atherosclerosis, cardiovasculardisease, impaired glucose tolerance, insulin resistance, hypertension,stroke, Syndrome X, Type I diabetes, Type II diabetes and lipoatrophicdiabetes. Diabetes-related complications to be treated by the methods ofthe invention include microangiopathic lesions, ocular lesions,retinopathy, neuropathy and renal lesions. Heart disease includes, butis not limited to, cardiac insufficiency, coronary insufficiency, andhigh blood pressure. Other metabolic-related disorders to be treated bycompounds of the invention include hyperlipidemia, hypertriglyceridemia,and hyperuricemia. Yet other metabolic-related diseases or disorders ofthe invention include cachexia, wasting, AIDS-related weight loss,cancer-related weight loss, neoplasia-related weight loss, anorexia, andbulimia. In preferred embodiments, GMG-3, GMG-4, Cluster 1, GMG-6A, orGMG-6B polypeptides in pharmaceutical compositions are used to modulatebody weight in healthy individuals for cosmetic reasons.

The invention also features a method of preventing or treatingmetabolic-related diseases and disorders comprising providing anindividual in need of such treatment with a compound identified byassays of the invention (described in Section VI of the PreferredEmbodiments of the Invention and in the Examples). Preferably thesecompounds antagonize or agonize effects of GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B polypeptides in cells in vitro, muscles ex vivo, or inanimal models. Alternatively, these compounds agonize or antagonize theeffects of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides onleptin and/or lipoprotein uptake and/or binding. Optionally, thesecompounds prevent the interaction, binding, or uptake of GMG-3, GMG-4,Cluster 1, GMG-6A, or GMG-6B polypeptides with LSR in vitro or in vivo.Preferably, the compound is provided to the individual in apharmaceutical composition that is preferably taken orally. Preferablythe individual is a mammal, and most preferably a human. In preferredembodiments, the metabolic-related disease or disorder is selected fromthe group consisting of obesity and metabolic-related diseases anddisorders such as atherosclerosis, heart disease, insulin resistance,hypertension, stroke, Syndrome X, Type I diabetes, Type II diabetes, andlipoatrophic diabetes. Diabetes-related complications to be treated bythe methods of the invention include microangiopathic lesions, ocularlesions, retinopathy, neuropathy and renal lesions. Heart diseaseincludes, but is not limited to, cardiac insufficiency, coronaryinsufficiency, and high blood pressure. Other metabolic-relateddisorders to be treated by compounds of the invention includehyperlipidemia, hypertriglyceridemia, and hyperuricemia.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to control blood glucose in some individuals, particularlythose with Type I diabetes, Type II diabetes, or insulin resistance, incombination with insulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to control body weight in some individuals, particularly thosewith Type I diabetes, Type II diabetes, or insulin resistance, incombination with insulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to control blood glucose in some individuals, particularlythose with Type I diabetes, Type II diabetes, or insulin resistance,alone, without combination of insulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to control body weight in some individuals, particularly thosewith Type II diabetes or insulin resistance, alone, without combinationof insulin therapy. In still a further preferred embodiment, the controlof body weight is due in part or in whole to a decrease in mass of 1)subcutaneous adipose tissue and/or 2) viseral (omental) adipose tissue.

In a further preferred embodiment, the present invention may be used incomplementary therapy, particularly in some individuals, particularlythose with Type I diabetes, Type II diabetes, or insulin resistance, toimprove their weight or glucose control in combination with an insulinsecretagogue or an insulin sensitising agent. Preferably, the insulinsecretagogue is 1,1-dimethyl-2-(2-morpholino phenyl)guanidine fumarate(BTS67582) or a sulphonylurea selected from tolbutamide, tolazamide,chlorpropamide, glibenclamide, glimepiride, glipizide, and glidazide.Preferably, the insulin sensitising agent is selected from metformin,ciglitazone, troglitazone and pioglitazone.

The present invention further provides a method of improving the bodyweight or glucose control of some individuals, particularly those withType I diabetes, Type II diabetes, or insulin resistance, alone, withoutan insulin secretagogue or an insulin sensitising agent.

In a further preferred embodiment, the present invention may beadministered either concomitantly or concurrently, with the insulinsecretagogue or insulin sensitising agent for example in the form ofseparate dosage units to be used simultaneously, separately orsequentially (either before or after the secretagogue or either beforeor after the sensitising agent). Accordingly, the present inventionfurther provides for a composition of pharmaceutical or physiologicallyacceptable composition and an oral insulin secretagogue or insulinsensitising agent as a combined preparation for simultaneous, separateor sequential use for the improvement of body weight or glucose controlin some individuals, particularly those with Type I diabetes, Type IIdiabetes, or insulin resistance.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition furtherprovides a method for the use as an insulin sensitiser.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to improve insulin sensitivity in some individuals,particularly those with Type I diabetes, Type II diabetes, or insulinresistance, in combination with insulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to improve insulin sensitivity in some individuals,particularly those with Type II diabetes or insulin resistance, withoutinsulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition furtherprovides a method for the use as an inhibitor of the progression fromimpaired glucose tolerance to insulin resistance.

IX. Ligands Interacting with GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6BPolypeptides.

For the purpose of the present invention, a Ligand means a molecule,such as a protein, a peptide, an antibody or any synthetic chemicalcompound capable of binding to a GMG-3, GMG-4, Cluster 1, GMG-6A, orGMG-6B protein or one of its fragments or variants or to modulate theexpression of the polynucleotide coding for GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B or a fragment or variant thereof.

In the Ligand screening method according to the present invention, abiological sample or a defined molecule to be tested as a putativeLigand of a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B protein isbrought into contact with the corresponding purified GMG-3, GMG-4,Cluster 1, GMG-6A, or GMG-6B protein, for example the correspondingpurified recombinant GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B proteinproduced by a recombinant cell host as described herein, in order toform a complex between this protein and the putative Ligand molecule tobe tested.

As an illustrative example, to study the interaction of a GMG-3, GMG-4,Cluster 1, GMG-6A, or GMG-6B protein, or a fragment comprising acontiguous span of at least 6 amino acids, preferably at least 8 to 10amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100amino acids of a polypeptide selected from the group consisting of ofSEQ ID NO: 2, 4, 6, 8, and 10, with drugs or small molecules, such asmolecules generated through combinatorial chemistry approaches, themicrodialysis coupled to HPLC method described by Wang et al. (1997) orthe affinity capillary electrophoresis method described by Bush et al.(1997), the disclosures of which are incorporated by reference, can beused.

In further methods, peptides, drugs, fatty acids, lipoproteins, or smallmolecules which interact with a GMG-3, GMG-4, Cluster 1, GMG-6A, orGMG-6B protein, or a fragment comprising a contiguous span of at least 6amino acids, preferably at least 8 to 10 amino acids, more preferably atleast 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of a polypeptideselected from the group consisting of sequences of SEQ ID NO: 2, 4, 6,8, or 10 may be identified using assays such as the following. Themolecule to be tested for binding is labelled with a detectable label,such as a fluorescent, radioactive, or enzymatic tag and placed incontact with immobilized GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bprotein, or a fragment thereof under conditions that permit specificbinding to occur. After removal of non-specifically bound molecules,bound molecules are detected using appropriate means.

Various candidate substances or molecules can be assayed for interactionwith a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide. Thesesubstances or molecules include, without being limited to, natural orsynthetic organic compounds or molecules of biological origin such aspolypeptides. When the candidate substance or molecule comprises apolypeptide, this polypeptide may be the resulting expression product ofa phage clone belonging to a phage-based random peptide library, oralternatively the polypeptide may be the resulting expression product ofa cDNA library cloned in a vector suitable for performing a two-hybridscreening assay.

A. Candidate Ligands Obtained by Affinity Chromatography.

Proteins or other molecules interacting with a GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B protein, or a fragment thereof comprising a contiguousspan of at least 6 amino acids, preferably at least 8 to 10 amino acids,more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acidsof a polypeptide selected from the group consisting of sequences of SEQID NO: 2, 4, 6, 8, and 10, can also be found using affinity columnswhich contain the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B protein, ora fragment thereof. The GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bprotein, or a fragment thereof, may be attached to the column usingconventional techniques including chemical coupling to a suitable columnmatrix such as agarose, Affi Gel®, or other matrices familiar to thoseof skill in art. In some embodiments of this method, the affinity columncontains chimeric proteins in which the GMG-3, GMG-4, Cluster 1, GMG-6A,or GMG-6B protein, or a fragment thereof, is fused to glutathion Stransferase (GST). A mixture of cellular proteins or pool of expressedproteins as described above is applied to the affinity column. Proteinsor other molecules interacting with the GMG-3, GMG-4, Cluster 1, GMG-6A,or GMG-6B protein, or a fragment thereof, attached to the column canthen be isolated and analyzed on 2-D electrophoresis gel as described inRamunsen et al. (1997), the disclosure of which is incorporated byreference. Alternatively, the proteins retained on the affinity columncan be purified by electrophoresis-based methods and sequenced. The samemethod can be used to isolate antibodies, to screen phage displayproducts, or to screen phage display human antibodies.

B. Candidate Ligands Obtained by Optical Biosensor Methods

Proteins interacting with a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bprotein, or a fragment comprising a contiguous span of at least 6 aminoacids, preferably at least 8 to 10 amino acids, more preferably at least12, 15, 20, 25, 30, 40, 50, or 100 amino acids of a polypeptide selectedfrom the group consisting of sequences of SEQ ID NO: 2, 4, 6, 8, or 10,can also be screened by using an Optical Biosensor as described inEdwards and Leatherbarrow (1997) and also in Szabo et al. (1995), thedisclosures of which are incorporated by reference. This techniquepermits the detection of interactions between molecules in real time,without the need of labelled molecules. This technique is based on thesurface plasmon resonance (SPR) phenomenon. Briefly, the candidateLigand molecule to be tested is attached to a surface (such as acarboxymethyl dextran matrix). A light beam is directed towards the sideof the surface that does not contain the sample to be tested and isreflected by said surface. The SPR phenomenon causes a decrease in theintensity of the reflected light with a specific association of angleand wavelength. The binding of candidate Ligand molecules cause a changein the refraction index on the surface, which change is detected as achange in the SPR signal. For screening of candidate Ligand molecules orsubstances that are able to interact with the GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B protein, or a fragment thereof, the GMG-3, GMG-4,Cluster 1, GMG-6A, or GMG-6B protein, or a fragment thereof, isimmobilized onto a surface. This surface comprises one side of a cellthrough which flows the candidate molecule to be assayed. The binding ofthe candidate molecule on the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bprotein, or a fragment thereof, is detected as a change of the SPRsignal. The candidate molecules tested may be proteins, peptides,carbohydrates, lipids, or small molecules generated by combinatorialchemistry. This technique may also be performed by immobilizingeukaryotic or prokaryotic cells or lipid vesicles exhibiting anendogenous or a recombinantly expressed GMG-3, GMG-4, Cluster 1, GMG-6A,or GMG-6B protein at their surface.

The main advantage of the method is that it allows the determination ofthe association rate between the GMG-3, GMG-4, Cluster 1, GMG-6A, orGMG-6B protein and molecules interacting with the GMG-3, GMG-4, Cluster1, GMG-6A, or GMG-6B protein. It is thus possible to select specificallyLigand molecules interacting with the GMG-3, GMG-4, Cluster 1, GMG-6A,or GMG-6B protein, or a fragment thereof, through strong or converselyweak association constants.

C. Candidate Ligands Obtained Through a Two-Hybrid Screening Assay.

The yeast two-hybrid system is designed to study protein-proteininteractions in vivo (Fields and Song, 1989), which disclosure is herebyincorporated by reference in its entirety, and relies upon the fusion ofa bait protein to the DNA binding domain of the yeast Gal4 protein. Thistechnique is also described in the U.S. Pat. No. 5,667,973 and the U.S.Pat. No. 5,283,173, the technical teachings of both patents being hereinincorporated by reference.

The general procedure of library screening by the two-hybrid assay maybe performed as described by Harper et al. (1993) or as described by Choet al. (1998) or also Fromont-Racine et al. (1997), which disclosuresare hereby incorporated by reference in their entireties.

The bait protein or polypeptide comprises, consists essentially of, orconsists of a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide ora fragment thereof comprising a contiguous span of at least 6 aminoacids, preferably at least 8 to 10 amino acids, more preferably at least12, 15, 20, 25, 30, 40, 50, or 100 amino acids of a polypeptide selectedfrom the group consisting of sequences of SEQ ID NO: 2, 4, 6, 8, and 10.

More precisely, the nucleotide sequence encoding the GMG-3, GMG-4,Cluster 1, GMG-6A, or GMG-6B polypeptide or a fragment or variantthereof is fused to a polynucleotide encoding the DNA binding domain ofthe GAL4 protein, the fused nucleotide sequence being inserted in asuitable expression vector, for example pAS2 or pM3.

Then, a human cDNA library is constructed in a specially designedvector, such that the human cDNA insert is fused to a nucleotidesequence in the vector that encodes the transcriptional domain of theGAL4 protein. Preferably, the vector used is the pACT vector. Thepolypeptides encoded by the nucleotide inserts of the human cDNA libraryare termed “prey” polypeptides.

A third vector contains a detectable marker gene, such as betagalactosidase gene or CAT gene that is placed under the control of aregulation sequence that is responsive to the binding of a complete Gal4protein containing both the transcriptional activation domain and theDNA binding domain. For example, the vector pG5EC may be used.

Two different yeast strains are also used. As an illustrative butnon-limiting example the two different yeast strains may be thefollowing:

Y190, the phenotype of which is (MATa, Leu2-3, 112 ura3-12, trp1-901,his3-D200, ade2-101, gal4Dgal180D URA3 GAL-LacZ, LYS GAL-HIS3, cyh^(r));

Y187, the phenotype of which is (MATa gal4 gal80his3 trp1-901 ade2-101ura3-52 leu2-3, -112 URA3 GAL-lacZmet⁻), which is the opposite matingtype of Y190.

Briefly, 20 μg of pAS2/GMG-3, pAS2/GMG-4, pAS2/Cluster 1, pAS2/GMG-6A,or pAS2/GMG-6B and 20 μg of pACT-cDNA library are co-transformed intoyeast strain Y190. The transformants are selected for growth on minimalmedia lacking histidine, leucine and tryptophan, but containing thehistidine synthesis inhibitor 3-AT (50 mM). Positive colonies arescreened for beta galactosidase by filter lift assay. The doublepositive colonies (His⁺, beta-gal⁺) are then grown on plates lackinghistidine, leucine, but containing tryptophan and cycloheximide (10mg/ml) to select for loss of pAS2/GMG-3, pAS2/GMG-4, pAS2/Cluster 1,pAS2/GMG-6A, or pAS2/GMG-6B plasmids but retention of pACT-cDNA libraryplasmids. The resulting Y190 strains are mated with Y187 strainsexpressing GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B or non-relatedcontrol proteins; such as cyclophilin B, lamin, or SNF1, as Gal4 fusionsas described by Harper et al. (1993) and by Bram et al. (1993), whichdisclosures are hereby incorporated by reference in their entireties,and screened for beta galactosidase by filter lift assay. Yeast clonesthat are beta gal- after mating with the control Gal4 fusions areconsidered false positives.

In another embodiment of the two-hybrid method according to theinvention, interaction between the GMG-3, GMG-4, Cluster 1, GMG-6A, orGMG-6B or a fragment or variant thereof with cellular proteins may beassessed using the Matchmaker Two Hybrid System 2 (Catalog No. K1604-1,Clontech). As described in the manual accompanying the kit, thedisclosure of which is incorporated herein by reference, nucleic acidsencoding the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B protein or aportion thereof, are inserted into an expression vector such that theyare in frame with DNA encoding the DNA binding domain of the yeasttranscriptional activator GAL4. A desired cDNA, preferably human cDNA,is inserted into a second expression vector such that they are in framewith DNA encoding the activation domain of GAL4. The two expressionplasmids are transformed into yeast and the yeast are plated onselection medium which selects for expression of selectable markers oneach of the expression vectors as well as GALA dependent expression ofthe HIS3 gene. Transformants capable of growing on medium lackinghistidine are screened for GAL4 dependent lacZ expression. Those cellsthat are positive in both the histidine selection and the lacZ assaycontain interaction between GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Band the protein or peptide encoded by the initially selected cDNAinsert.

X. Assays for Identifying Modulators of GMG-3, GMG-4, Cluster 1, GMG-6A,or GMG-6B Polypeptide Activity

The invention features methods of screening for one or more compoundsthat modulate the activity of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bin cells, which includes providing potential compounds to be tested tothe cells. Exemplary assays that may be used are described in theExamples section. To these assays would be added compounds to be testedfor their inhibitory or stimulatory activity as compared to the effectsof GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides alone. Otherassays in which an effect is observed based on the addition of GMG-3,GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides can also be used toscreen for modulators of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bpolypeptide activity or effects of the presence of GMG-3, GMG-4, Cluster1, GMG-6A, or GMG-6B polypeptides on cells. The essential step is toapply an unknown compound and then to monitor an assay for a change fromwhat is seen when only GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bpolypeptides are applied to the cell. A change is defined as somethingthat is significantly different in the presence of the compound plusGMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide compared toGMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide alone. In thiscase, significantly different would be an “increase” or a “decrease” ina measurable effect of at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, or 75%.

The term “modulation” as used herein refers to a measurable change in anactivity. Examples include, but are not limited to, lipolysis stimulatedreceptor (LSR) modulation, leptin modulation, lipoprotein modulation,plasma FFA levels, FFA oxidation, TG levels, glucose levels, and weight.These effects can be in vitro or preferably in vivo. Modulation of anactivity can be either an increase or a decrease in the activity. Thus,LSR activity can be increased or decreased, leptin activity can beincreased or decreased, and lipoprotein activity can be increased ordecreased. Similarly, FFA, TG, glucose levels and weight can beincreased or decreased in vivo Free Fatty Acid oxidation can beincreased or decreased in vivo or ex vivo.

By “LSR” activity is meant expression of LSR on the surface of the cell,or in a particular conformation, as well as its ability to bind, uptake,and degrade leptin and lipoprotein. By “leptin” activity is meant itsbinding, uptake and degradation by LSR, as well as its transport acrossa blood brain barrier, and potentially these occurrences where LSR isnot necessarily the mediating factor or the only mediating factor.Similarly, by “lipoprotein” activity is meant its binding, uptake anddegradation by LSR, as well as these occurrences where LSR is notnecessarily the mediating factor or the only mediating factor. Exemplaryassays are provided in the Examples. These assay and other comparableassays can be used to determine/identify compounds that modulate GMG-3,GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide activity. In some casesit may be important to identify compounds that modulate some but not allof the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptideactivities, although preferably all activities are modified.

The term “increasing” as used herein refers to the ability of a compoundto increase the activity of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bpolypeptides in some measurable way compared to the effect of GMG-3,GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides in its absence. As aresult of the presence of the compound leptin binding and/or uptakemight increase, for example, as compared to controls in the presence ofthe GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide alone.Preferably, an increase in activity is at least 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, or 75% compared to the level of activity in thepresence of the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide.Preferably said GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptideis GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide fragmentcomprising all or part of the C-terminal globular C1q homology domain.

Similarly, the term “decreasing” as used herein refers to the ability ofa compound to decrease an activity in some measurable way compared tothe effect of a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptidein its absence. For example, the presence of the compound decreases theplasma concentrations of FFA, TG, and glucose in mice. Also as a resultof the presence of a compound leptin binding and/or uptake mightdecrease, for example, as compared to controls in the presence of theGMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides alone.Preferably, an decrease in activity is at least 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, or 75% as compared to the level of activity inthe presence of the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bpolypeptides alone. Preferably said GMG-3, GMG-4, Cluster 1, GMG-6A, orGMG-6B polypeptide is GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bpolypeptide fragment comprising all or part of the C-terminal globularC1q homology domain.

The invention features a method for identifying a potential compound todecrease body mass in individuals in need of decreasing body masscomprising: a) contacting a cell with a GMG-3, GMG-4, Cluster 1, GMG-6A,or GMG-6B polypeptide and a candidate compound; b) detecting a resultselected from the group consisting of LSR modulation, leptin modulation,increase in glucose uptake or oxidation, decrease in blood lipid ortriglyceride levels, increase in lipoprotein binding, uptake ordegradation; FFA oxidation increase; and c) wherein said resultidentifies said potential compound if said result differs from saidresult when said cell is contacted with the GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B polypeptide alone.

Alternatively, the invention features a method for identifying apotential compound to increase body mass in individuals in need ofincreasing body mass comprising: a) contacting a cell with a GMG-3,GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide and a candidatecompound; b) detecting a result selected from the group consisting ofLSR modulation, leptin modulation, decrease in glucose uptake oroxidation, increase in blood lipid or triglyceride levels, decrease inlipoprotein binding, uptake or degradation; FFA oxidation decrease; andc) wherein said result identifies said potential compound if said resultdiffers from said result when said cell is contacted with the GMG-3,GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide alone.

In still other preferred embodiments, said potential compound isselected from the group consisting of peptides, peptide libraries,non-peptide libraries, peptoids, fatty acids, lipoproteins, medicaments,antibodies, small molecules, proteases and protease inhibitors.

XI. Epitopes and Antibody Fusions

A preferred embodiment of the present invention is directed toepitope-bearing polypeptides and epitope-bearing polypeptide fragments.These epitopes may be “antigenic epitopes” or both an “antigenicepitope” and an “immunogenic epitope”. An “immunogenic epitope” isdefined as a part of a protein that elicits an antibody response in vivowhen the polypeptide is the immunogen. On the other hand, a region ofpolypeptide to which an antibody binds is defined as an “antigenicdeterminant” or “antigenic epitope.” The number of immunogenic epitopesof a protein generally is less than the number of antigenic epitopes.See, e.g., Geysen, et al. (1983) Proc Natl Acad Sci USA 81:3998-4002. Itis particularly noted that although a particular epitope may not beimmunogenic, it is nonetheless useful since antibodies can be made invitro to any epitope.

An epitope can comprise as few as 3 amino acids in a spatialconformation which is unique to the epitope. Generally an epitopeconsists of at least 6 such amino acids, and more often at least 8-10such amino acids. In preferred embodiment, antigenic epitopes comprise anumber of amino acids that is any integer between 3 and 50. Fragmentswhich function as epitopes may be produced by any conventional means.See, e.g., Houghten, R. A., Proc Natl Acad Sci USA 82:5131-5135 (1985),further described in U.S. Pat. No. 4,631,211. Methods for determiningthe amino acids which make up an immunogenic epitope include x-raycrystallography, 2-dimensional nuclear magnetic resonance, and epitopemapping, e.g., the Pepscan method described by H. Mario Geysen et al.(1984); Proc. Natl. Acad. Sci. U.S.A. 81:3998-4002; PCT Publication No.WO 84/03564; and PCT Publication No. WO 84/03506. Another example is thealgorithm of Jameson and Wolf, Comp. Appl. Biosci. 4:181-186 (1988)(said references incorporated by reference in their entireties). TheJameson-Wolf antigenic analysis, for example, may be performed using thecomputer program PROTEAN, using default parameters (Version 4.0 Windows,DNASTAR, Inc., 1228 South Park Street Madison, Wis.).

The epitope-bearing fragments of the present invention preferablycomprise 6 to 50 amino acids (i.e. any integer between 6 and 50,inclusive) of a polypeptide of the present invention. Also, included inthe present invention are antigenic fragments between the integers of 6and the full-length sequence of the sequence listing. All combinationsof sequences between the integers of 6 and the full-length sequence of apolypeptide of the present invention are included. The epitope-bearingfragments may be specified by either the number of contiguous amino acidresidues (as a sub-genus) or by specific N-terminal and C-terminalpositions (as species) as described above for the polypeptide fragmentsof the present invention. Any number of epitope-bearing fragments of thepresent invention may also be excluded in the same manner.

Antigenic epitopes are useful, for example, to raise antibodies,including monoclonal antibodies that specifically bind the epitope (See,Wilson et al., 1984; and Sutcliffe, J. G. et al., 1983). The antibodiesare then used in various techniques such as diagnostic and tissue/cellidentification techniques, as described herein, and in purificationmethods.

Similarly, immunogenic epitopes can be used to induce antibodiesaccording to methods well known in the art (See, Sutcliffe et al.,supra; Wilson et al., supra; Chow, M. et al.; (1985) and Bittle, F. J.et al., (1985). A preferred immunogenic epitope includes thepolypeptides of the sequence listing. The immunogenic epitopes may bepresented together with a carrier protein, such as an albumin, to ananimal system (such as rabbit or mouse) if necessary. Immunogenicepitopes comprising as few as 8 to 10 amino acids have been shown to besufficient to raise antibodies capable of binding to, at the very least,linear epitopes in a denatured polypeptide (e.g., in Western blotting.).

Epitope-bearing polypeptides of the present invention are used to induceantibodies according to methods well known in the art including, but notlimited to, in vivo immunization, in vitro immunization, and phagedisplay methods (See, e.g., Sutcliffe, et al., supra; Wilson, et al.,supra, and Bittle, et al., 1985). If in vivo immunization is used,animals may be immunized with free peptide; however, anti-peptideantibody titer may be boosted by coupling of the peptide to amacromolecular carrier, such as keyhole limpet hemacyanin (KLH) ortetanus toxoid. For instance, peptides containing cysteine residues maybe coupled to a carrier using a linker such as-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptidesmay be coupled to carriers using a more general linking agent such asglutaraldehyde. Animals such as rabbits, rats and mice are immunizedwith either free or carrier-coupled peptides, for instance, byintraperitoneal and/or intradermal injection of emulsions containingabout 100 μgs of peptide or carrier protein and Freund's adjuvant.Several booster injections may be needed, for instance, at intervals ofabout two weeks, to provide a useful titer of anti-peptide antibody,which can be detected, for example, by ELISA assay using free peptideadsorbed to a solid surface. The titer of anti-peptide antibodies inserum from an immunized animal may be increased by selection ofanti-peptide antibodies, for instance, by adsorption to the peptide on asolid support and elution of the selected antibodies according tomethods well known in the art.

As one of skill in the art will appreciate, and discussed above, thepolypeptides of the present invention including, but not limited to,polypeptides comprising an immunogenic or antigenic epitope can be fusedto heterologous polypeptide sequences. For example, the polypeptides ofthe present invention may be fused with the constant region comprisingportions of immunoglobulins (IgA, IgE, IgG, IgM), or portions of theconstant region (CH1, CH2, CH3, any combination thereof including bothentire domains and portions thereof) resulting in chimeric polypeptides.These fusion proteins facilitate purification, and show an increasedhalf-life in vivo. This has been shown, e.g., for chimeric proteinsconsisting of the first two domains of the human CD4-polypeptide andvarious domains of the constant regions of the heavy or light chains ofmammalian immunoglobulins (See, e.g., EPA 0,394,827; and Traunecker etal., 1988). Fusion proteins that have a disulfide-linked dimericstructure due to the IgG portion can also be more efficient in bindingand neutralizing other molecules than monomeric polypeptides orfragments thereof alone (See, e.g., Fountoulakis et al., 1995). Nucleicacids encoding the above epitopes can also be recombined with a gene ofinterest as an epitope tag to aid in detection and purification of theexpressed polypeptide.

Additional fusion proteins of the invention may be generated through thetechniques of gene-shuffling, motif-shuffling, exon-shuffling, orcodon-shuffling (collectively referred to as “DNA shuffling”). DNAshuffling may be employed to modulate the activities of polypeptides ofthe present invention thereby effectively generating agonists andantagonists of the polypeptides. See, for example, U.S. Pat. Nos.5,605,793; 5,811,238; 5,834,252; 5,837,458; and Patten, P. A., et al.,(1997); Harayama, S., (1998); Hansson, L. O., et al (1999); and Lorenzo,M. M. and Blasco, R., (1998). (Each of these documents are herebyincorporated by reference). In one embodiment, one or more components,motifs, sections, parts, domains, fragments, etc., of codingpolynucleotides of the invention, or the polypeptides encoded therebymay be recombined with one or more components, motifs, sections, parts,domains, fragments, etc. of one or more heterologous molecules.

Antibodies

The present invention further relates to antibodies and T-cell antigenreceptors (TCR) that specifically bind the polypeptides, and morespecifically, the epitopes of the polypeptides of the present invention.The antibodies of the present invention include IgG (including IgG1,IgG2, IgG3, and IgG4), IgA (including IgA1 and IgA2), IgD, IgE, or IgM,and IgY. As used herein, the term “antibody” (Ab) is meant to includewhole antibodies, including single-chain whole antibodies, and antigenbinding fragments thereof. In a preferred embodiment the antibodies arehuman antigen binding antibody fragments of the present inventioninclude, but are not limited to, Fab, Fab′ F(ab)2 and F(ab′)2, Fd,single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs(sdFv) and fragments comprising either a V_(L) or V_(H) domain. Theantibodies may be from any animal origin including birds and mammals.Preferably, the antibodies are human, murine, rabbit, goat, guinea pig,camel, horse, or chicken.

Antigen-binding antibody fragments, including single-chain antibodies,may comprise the variable region(s) alone or in combination with theentire or partial of the following: hinge region, CH1, CH2, and CH3domains. Also included in the invention are any combinations of variableregion(s) and hinge region, CH1, CH2, and CH3 domains. The presentinvention further includes chimeric, humanized, and human monoclonal andpolyclonal antibodies, which specifically bind the polypeptides of thepresent invention. The present invention further includes antibodiesthat are anti-idiotypic to the antibodies of the present invention.

The antibodies of the present invention may be monospecific, bispecific,and trispecific or have greater multispecificity. Multispecificantibodies may be specific for different epitopes of a polypeptide ofthe present invention or may be specific for both a polypeptide of thepresent invention as well as for heterologous compositions, such as aheterologous polypeptide or solid support material. See, e.g., WO93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, A. et al. (1991);U.S. Pat. Nos. 5,573,920, 4,474,893, 5,601,819, 4,714,681, 4,925,648;Kostelny, S. A. et al. (1992).

Antibodies of the present invention may be described or specified interms of the epitope(s) or epitope-bearing portion(s) of a polypeptideof the present invention, which are recognized or specifically bound bythe antibody. In the case of proteins of the present invention secretedproteins, the antibodies may specifically bind a full-length proteinencoded by a nucleic acid of the present invention, a mature protein(i.e., the protein generated by cleavage of the signal peptide) encodedby a nucleic acid of the present invention, a signal peptide encoded bya nucleic acid of the present invention, or any other polypeptide of thepresent invention. Therefore, the epitope(s) or epitope bearingpolypeptide portion(s) may be specified as described herein, e.g., byN-terminal and C-terminal positions, by size in contiguous amino acidresidues, or otherwise described herein. Antibodies that specificallybind any epitope or polypeptide of the present invention may also beexcluded as individual species. Therefore, the present inventionincludes antibodies that specifically bind specified polypeptides of thepresent invention, and allows for the exclusion of the same.

Antibodies of the present invention may also be described or specifiedin terms of their cross-reactivity. Antibodies that do not specificallybind any other analog, ortholog, or homolog of the polypeptides of thepresent invention are included. Antibodies that do not bind polypeptideswith less than 95%, less than 90%, less than 85%, less than 80%, lessthan 75%, less than 70%, less than 65%, less than 60%, less than 55%,and less than 50% identity (as calculated using methods known in the artand described herein, eg., using FASTDB and the parameters set forthherein) to a polypeptide of the present invention are also included inthe present invention. Further included in the present invention areantibodies, which only bind polypeptides encoded by polynucleotides,which hybridize to a polynucleotide of the present invention understringent hybridization conditions (as described herein). Antibodies ofthe present invention may also be described or specified in terms oftheir binding affinity. Preferred binding affinities include those witha dissociation constant or Kd value less than 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M,5×10⁻¹¹ M, 10⁻¹¹M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M,10⁻¹⁴ M, 5×10⁻¹⁵ M, and 10⁻¹⁵ M.

Antibodies of the present invention have uses that include, but are notlimited to, methods known in the art to purify, detect, and target thepolypeptides of the present invention including both in vitro and invivo diagnostic and therapeutic methods. For example, the antibodieshave use in immunoassays for qualitatively and quantitatively measuringlevels of the polypeptides of the present invention in biologicalsamples (See, e.g., Harlow et al., 1988).

The antibodies of the present invention may be used either alone or incombination with other compositions. The antibodies may further berecombinantly fused to a heterologous polypeptide at the N- orC-terminus or chemically conjugated (including covalent and non-covalentconjugations) to polypeptides or other compositions. For example,antibodies of the present invention may be recombinantly fused orconjugated to molecules useful as labels in detection assays andeffector molecules such as heterologous polypeptides, drugs, or toxins.See, e.g., WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No.5,314,995; and EP 0 396 387.

The antibodies of the present invention may be prepared by any suitablemethod known in the art. For example, a polypeptide of the presentinvention or an antigenic fragment thereof can be administered to ananimal in order to induce the production of sera containing polyclonalantibodies. The term “monoclonal antibody” is not limited to antibodiesproduced through hybridoma technology. The term “antibody” refers to apolypeptide or group of polypeptides which are comprised of at least onebinding domain, where a binding domain is formed from the folding ofvariable domains of an antibody molecule to form three-dimensionalbinding spaces with an internal surface shape and charge distributioncomplementary to the features of an antigenic determinant of an antigen,which allows an immunological reaction with the antigen. The term“monoclonal antibody” refers to an antibody that is derived from asingle clone, including eukaryotic, prokaryotic, or phage clone, and notthe method by which it is produced. Monoclonal antibodies can beprepared using a wide variety of techniques known in the art includingthe use of hybridoma, recombinant, and phage display technology.

Hybridoma techniques include those known in the art (See, e.g., Harlowet al. 1988); Hammerling, et al, 1981) (said references incorporated byreference in their entireties). Fab and F(ab′)2 fragments may beproduced, for example, from hybridoma-produced antibodies by proteolyticcleavage, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments).

Alternatively, antibodies of the present invention can be producedthrough the application of recombinant DNA technology or throughsynthetic chemistry using methods known in the art. For example, theantibodies of the present invention can be prepared using various phagedisplay methods known in the art. In phage display methods, functionalantibody domains are displayed on the surface of a phage particle, whichcarries polynucleotide sequences encoding them. Phage with a desiredbinding property are selected from a repertoire or combinatorialantibody library (e.g. human or murine) by selecting directly withantigen, typically antigen bound or captured to a solid surface or bead.Phage used in these methods are typically filamentous phage including fdand M13 with Fab, Fv or disulfide stabilized Fv antibody domainsrecombinantly fused to either the phage gene III or gene VIII protein.Examples of phage display methods that can be used to make theantibodies of the present invention include those disclosed in BrinkmanU. et al. (1995); Ames, R. S. et al. (1995); Kettleborough, C. A. et al.(1994); Persic, L. et al. (1997); Burton, D. R. et al. (1994);PCT/GB91/01134; WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426,5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047,5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727 and 5,733,743.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired hostincluding mammalian cells, insect cells, plant cells, yeast, andbacteria. For example, techniques to recombinantly produce Fab, Fab′F(ab)2 and F(ab′)2 fragments can also be employed using methods known inthe art such as those disclosed in WO 92/22324; Mullinax, R. L. et al.(1992); and Sawai, H. et al. (1995); and Better, M. et al. (1988).

Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al. (1991); Shu, L. et al. (1993); and Skerra, A.et al. (1988). For some uses, including in vivo use of antibodies inhumans and in vitro detection assays, it may be preferable to usechimeric, humanized, or human antibodies. Methods for producing chimericantibodies are known in the art. See e.g., Morrison, (1985); Oi et al.,(1986); Gillies, S. D. et al. (1989); and U.S. Pat. No. 5,807,715.Antibodies can be humanized using a variety of techniques includingCDR-grafting (EP 0 239 400; WO 91/09967; U.S. Pat. Nos. 5,530,101; and5,585,089), veneering or resurfacing, (EP 0 592 106; EP 0 519 596;Padlan E. A., 1991; Studnicka G. M. et al., 1994; Roguska M. A. et al.,1994), and chain shuffling (U.S. Pat. No. 5,565,332). Human antibodiescan be made by a variety of methods known in the art including phagedisplay methods described above. See also, U.S. Pat. Nos. 4,444,887,4,716,111, 5,545,806, and 5,814,318; WO 98/46645; WO 98/50433; WO98/24893; WO 96/34096; WO 96/33735; and WO 91/10741.

Further included in the present invention are antibodies recombinantlyfused or chemically conjugated (including both covalently andnon-covalently conjugations) to a polypeptide of the present invention.The antibodies may be specific for antigens other than polypeptides ofthe present invention. For example, antibodies may be used to target thepolypeptides of the present invention to particular cell types, eitherin vitro or in vivo, by fusing or conjugating the polypeptides of thepresent invention to antibodies specific for particular cell surfacereceptors. Antibodies fused or conjugated to the polypeptides of thepresent invention may also be used in in vitro immunoassays andpurification methods using methods known in the art (See e.g., Harbor etal. supra; WO 93/21232; EP 0 439 095; Naramura, M. et al. 0.1994; U.S.Pat. No. 5,474,981; Gillies, S. O. et al., 1992; Fell, H. P. et al.,1991).

The present invention further includes compositions comprising thepolypeptides of the present invention fused or conjugated to antibodydomains other than the variable regions. For example, the polypeptidesof the present invention may be fused or conjugated to an antibody Fcregion, or portion thereof. The antibody portion fused to a polypeptideof the present invention may comprise the hinge region, CH1 domain, CH2domain, and CH3 domain or any combination of whole domains or portionsthereof. The polypeptides of the present invention may be fused orconjugated to the above antibody portions to increase the in vivohalf-life of the polypeptides or for use in immunoassays using methodsknown in the art. The polypeptides may also be fused or conjugated tothe above antibody portions to form multimers. For example, Fc portionsfused to the polypeptides of the present invention can form dimersthrough disulfide bonding between the Fc portions. Higher multimericforms can be made by fusing the polypeptides to portions of IgA and IgM.Methods for fusing or conjugating the polypeptides of the presentinvention to antibody portions are known in the art. See e.g., U.S. Pat.Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,112,946;EP 0 307 434, EP 0 367 166; WO 96/04388, WO 91/06570; Ashkenazi, A. etal. (1991); Zheng, X. X. et al. (1995); and Vil, H. et al. (1992).

The invention further relates to antibodies that act as agonists orantagonists of the polypeptides of the present invention. For example,the present invention includes antibodies that disrupt thereceptor/ligand interactions with the polypeptides of the inventioneither partially or fully. Included are both receptor-specificantibodies and ligand-specific antibodies. Included arereceptor-specific antibodies, which do not prevent ligand binding butprevent receptor activation. Receptor activation (i.e., signaling) maybe determined by techniques described herein or otherwise known in theart. Also include are receptor-specific antibodies which both preventligand binding and receptor activation. Likewise, included areneutralizing antibodies that bind the ligand and prevent binding of theligand to the receptor, as well as antibodies that bind the ligand,thereby preventing receptor activation, but do not prevent the ligandfrom binding the receptor. Further included are antibodies that activatethe receptor. These antibodies may act as agonists for either all orless than all of the biological activities affected by ligand-mediatedreceptor activation. The antibodies may be specified as agonists orantagonists for biological activities comprising specific activitiesdisclosed herein. The above antibody agonists can be made using methodsknown in the art. See e.g., WO 96/40281; U.S. Pat. No. 5,811,097; Deng,B. et al. (1998); Chen, Z. et al. (1998); Harrop, J. A. et al. (1998);Zhu, Z. et al. (1998); Yoon, D. Y. et al. (1998); Prat, M. et al. (1998)J.; Pitard, V. et al. (1997); Liautard, J. et al. (1997); Carlson, N. G.et al. (1997) J.; Taryman, R. E. et al. (1995); Muller, Y. A. et al.(1998); Bartunek, P. et al. (1996).

As discussed above, antibodies of the polypeptides of the invention can,in turn, be utilized to generate anti-idiotypic antibodies that “mimic”polypeptides of the invention using techniques well known to thoseskilled in the art (See, e.g. Greenspan and Bona (1989); and Nissinoff(1991). For example, antibodies which bind to and competitively inhibitpolypeptide multimerization or binding of a polypeptide of the inventionto ligand can be used to generate anti-idiotypes that “mimic” thepolypeptide multimerization or binding domain and, as a consequence,bind to and neutralize polypeptide or its ligand. Such neutralizationanti-idiotypic antibodies can be used to bind a polypeptide of theinvention or to bind its ligands/receptors, and therby block itsbiological activity,

The invention also concerns a purified or isolated antibody capable ofspecifically binding to a mutated full length or mature polypeptide ofthe present invention or to a fragment or variant thereof comprising anepitope of the mutated polypeptide. In another preferred embodiment, thepresent invention concerns an antibody capable of binding to apolypeptide comprising at least 10 consecutive amino acids of apolypeptide of the present invention and including at least one of theamino acids which can be encoded by the trait causing mutations.

Non-human animals or mammals, whether wild-type or transgenic, whichexpress a different species of a polypeptide of the present inventionthan the one to which antibody binding is desired, and animals which donot express a polypeptide of the present invention (i.e. a knockoutanimal) are particularly useful for preparing antibodies. Gene knock outanimals will recognize all or most of the exposed regions of apolypeptide of the present invention as foreign antigens, and thereforeproduce antibodies with a wider array of epitopes. Moreover, smallerpolypeptides with only 10 to 30 amino acids may be useful in obtainingspecific binding to any one of the polypeptides of the presentinvention. In addition, the humoral immune system of animals thatproduce a species of a polypeptide of the present invention thatresembles the antigenic sequence will preferentially recognize thedifferences between the animal's native polypeptide species and theantigen sequence, and produce antibodies to these unique sites in theantigen sequence. Such a technique will be particularly useful inobtaining antibodies that specifically bind to any one of thepolypeptides of the present invention.

Antibody preparations prepared according to either protocol are usefulin quantitative immunoassays which determine concentrations ofantigen-bearing substances in biological samples; they are also usedsemi-quantitatively or qualitatively to identify the presence of antigenin a biological sample. The antibodies may also be used in therapeuticcompositions for killing cells expressing the protein or reducing thelevels of the protein in the body.

The antibodies of the invention may be labelled by any one of theradioactive, fluorescent or enzymatic labels known in the art.

Consequently, the invention is also directed to a method for detectingspecifically the presence of a polypeptide of the present inventionaccording to the invention in a biological sample, said methodcomprising the following steps:

a) obtaining a biological sample suspected of containing a polypeptideof the present invention;

b) contacting the biological sample with a polyclonal or monoclonalantibody that specifically binds a polypeptide of the present inventionunder conditions suitable for antigen-antibody binding; and

c) detecting the antigen-antibody complex formed.

The invention also concerns a diagnostic kit for detecting in vitro thepresence of a polypeptide of the present invention in a biologicalsample, wherein said kit comprises:

a) a polyclonal or monoclonal antibody that specifically binds apolypeptide of the present invention, optionally labelled;

b) a reagent allowing the detection of the antigen-antibody complexesformed, said reagent carrying optionally a label, or being able to berecognized itself by a labelled reagent, more particularly in the casewhen the above-mentioned monoclonal or polyclonal antibody is notlabelled by itself.

A. Monoclonal Antibody Production by Hybridoma Fusion

Monoclonal antibody to epitopes of any of the peptides identified andisolated as described can be prepared from murine hybridomas accordingto the classical method of Kohler, G. and Milstein, C., Nature 256:495(1975) or derivative methods thereof. Briefly, a mouse is repetitivelyinoculated with a few micrograms of the selected protein or peptidesderived therefrom over a period of a few weeks. The mouse is thensacrificed, and the antibody producing cells of the spleen isolated. Thespleen cells are fused by means of polyethylene glycol with mousemyeloma cells, and the excess unfused cells destroyed by growth of thesystem on selective media comprising aminopterin (HAT media). Thesuccessfully fused cells are diluted and aliquots of the dilution placedin wells of a microtiter plate where growth of the culture is continued.Antibody-producing clones are identified by detection of antibody in thesupernatant fluid of the wells by immunoassay procedures, such as Elisa,as originally described by Engvall, E., Meth Enzymol 70:419 (1980), andderivative methods thereof. Selected positive clones can be expanded andtheir monoclonal antibody product harvested for use. Detailed proceduresfor monoclonal antibody production are described in Davis, L. et al.Basic Methods in Molecular Biology Elsevier, New York. Section 21-2.

In a preferred embodiment, said monoclonal antibody is specific for aGMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide or polypeptidefragment comprising all or part of the C-terminal globular C1q homologydomain. In preferred embodiments, said polypeptide fragment comprises,consists essentially of, or consists of, at least 6 consecutive aminoacids and not more than 333 consecutive amino acids of SEQ ID NO: 2 or4, preferably wherein said polypeptide fragment is comprised of one ormore of amino acids 227, 228, 229, 230, 231, 232, 233, 234, 235, 236,237, 238, 239, 240, 241, 242, 243, 244, 245, 245, 247, 248, 249, 250,251, 252, or 253, and more preferably wherein said polypeptide fragmentis comprised of the sequence TVFSRNVQVSLV (amino acids 256-267 of SEQ IDNO: 2 or 4) or QVTGGERFNGLFAD (amino acids 304-317 of SEQ ID NO: 2 or4); or at least 6 and not more than 225 consecutive amino acids of SEQID NO: 6, preferably wherein said polypeptide fragment is comprised ofone or more of amino acids 119, 120, 121, 122, 123, 124, 125, 126, 127,128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144, or 145, and more preferably wherein said polypeptidefragment is comprised of the sequence TVFSRNVQVSLV (amino acids 148-159of SEQ ID NO: 6) or QVTGGERFNGLFAD (amino acids 196-209 of SEQ ID NO:6); at least 6 consecutive amino acids and not more than 330 consecutiveamino acids of SEQ ID NO: 8, preferably wherein said polypeptidefragment is comprised of one or more of amino acids 224, 225, 226, 227,228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241,242, 243, 244, 245, 246, 247, 248, 249, or 250, and more preferablywherein said polypeptide fragment is comprised of the sequenceTVFSRNVQVSLV (amino acids 253-264 of SEQ ID NO: 8) or QVTGGERFNGLFAD(amino acids 301-314 of SEQ ID NO: 8); or at least 6 and not more than323 consecutive amino acids of SEQ ID NO: 10, preferably wherein saidpolypeptide fragment is comprised of one or more of amino acids 217,218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231,232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, or 243, and morepreferably wherein said polypeptide fragment is comprised of thesequence TVFSRNVQVSLV (amino acids 246-257 of SEQ ID NO: 10) orQVTGGERFNGLFAD (amino acids 294-307 of SEQ ID NO: 10).

B. Polyclonal Antibody Production by Immunization

Polyclonal antiserum containing antibodies to heterogenous epitopes of asingle protein can be prepared by immunizing suitable animals with theexpressed protein or peptides derived therefrom described above, whichcan be unmodified or modified to enhance immunogenicity. Effectivepolyclonal antibody production is affected by many factors related bothto the antigen and the host species. For example, small molecules tendto be less immunogenic than others and may require the use of carriersand adjuvant. Also, host animals vary in response to site ofinoculations and dose, with both inadequate or excessive doses ofantigen resulting in low titer antisera. Small doses (ng level) ofantigen administered at multiple intradermal sites appears to be mostreliable. An effective immunization protocol for rabbits can be found inVaitukaitis, J. et al. J. Clin. Endocrinol. Metab. 33:988-991 (1971).

Booster injections can be given at regular intervals, and antiserumharvested when antibody titer thereof, as determinedsemi-quantitatively, for example, by double immunodiffusion in agaragainst known concentrations of the antigen, begins to fall. See, forexample, Ouchterlony, O. et al., Chap. 19 in: Handbook of ExperimentalImmunology D. Wier (ed) Blackwell (1973). Plateau concentration ofantibody is usually in the range of 0.1 to 0.2 mg/ml of serum (about 12□M). Affinity of the antisera for the antigen is determined by preparingcompetitive binding curves, as described, for example, by Fisher, D.,Chap. 42 in: Manual of Clinical Immunology, 2d Ed. (Rose and Friedman,Eds.) Amer. Soc. For Microbiol., Washington, D.C. (1980).

In a preferred embodiment, said polyclonal antibody is specific for aGMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide or polypeptidefragment comprising all or part of the C-terminal globular C1q homologydomain. In preferred embodiments, said polypeptide fragment comprises,consists essentially of, or consists of, at least 6 consecutive aminoacids and not more than 333 consecutive amino acids of SEQ ID NO: 2 or4, preferably wherein said polypeptide fragment is comprised of one ormore of amino acids 227, 228, 229, 230, 231, 232, 233, 234, 235, 236,237, 238, 239, 240, 241, 242, 243, 244, 245, 245, 247, 248, 249, 250,251, 252, or 253, and more preferably wherein said polypeptide fragmentis comprised of the sequence TVFSRNVQVSLV (amino acids 256-267 of SEQ IDNO: 2 or 4) or QVTGGERFNGLFAD (amino acids 304-317 of SEQ ID NO: 2 or4); or at least 6 and not more than 225 consecutive amino acids of SEQID NO: 6, preferably wherein said polypeptide fragment is comprised ofone or more of amino acids 119, 120, 121, 122, 123, 124, 125, 126, 127,128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144, or 145, and more preferably wherein said polypeptidefragment is comprised of the sequence TVFSRNVQVSLV (amino acids 148-159of SEQ ID NO: 6) or QVTGGERFNGLFAD (amino acids 196-209 of SEQ ID NO:6); at least 6 consecutive amino acids and not more than 330 consecutiveamino acids of SEQ ID NO: 8, preferably wherein said polypeptidefragment is comprised of one or more of amino acids 224, 225, 226, 227,228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241,242, 243, 244, 245, 246, 247, 248, 249, or 250, and more preferablywherein said polypeptide fragment is comprised of the sequenceTVFSRNVQVSLV (amino acids 253-264 of SEQ ID NO: 8) or QVTGGERFNGLFAD(amino acids 301-314 of SEQ ID NO: 8); or at least 6 and not more than323 consecutive amino acids of SEQ ID NO: 10, preferably wherein saidpolypeptide fragment is comprised of one or more of amino acids 217,218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231,232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, or 243, and morepreferably wherein said polypeptide fragment is comprised of thesequence TVFSRNVQVSLV (amino acids 246-257 of SEQ ID NO: 10) orQVTGGERFNGLFAD (amino acids 294-307 of SEQ ID NO: 10).

Antibody preparations prepared according to either protocol are usefulin quantitative immunoassays which determine concentrations ofantigen-bearing substances in biological samples; they are also usedsemi-quantitatively or qualitatively to identify the presence of antigenin a biological sample. The antibodies may also be used in therapeuticcompositions for killing cells expressing the protein or reducing thelevels of the protein in the body.

XII. Identifying One or More Cell Types Expressing a Cell SurfaceReceptor for GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B Polypeptide

The invention features methods of identifying one or more cell typesexpressing a cell surface receptor for GMG-3, GMG-4, Cluster 1, GMG-6A,or GMG-6B polypeptide comprised of contacting said cell type withlabelled GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide andmeasuring the amount of said polypeptide bound. Preferably said GMG-3,GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide comprises all or part ofthe C-terminal globular C1q homology domain and has lipid partitioning,lipid metabolism, or insulin-like activities. Preferably said labelledGMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide is selected frombut not restricted to fluorescein-coupled GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B or biotin coupled GMG-3, GMG-4, Cluster 1, GMG-6A, orGMG-6B. Bound fluorescein-coupled GMG-3, GMG-4, Cluster 1, GMG-6A, orGMG-6B is detected directly by FACS. Bound biotin-coupled GMG-3, GMG-4,Cluster 1, GMG-6A, or GMG-6B is detected by FACS after secondary bindingof phycoerythrin-coupled streptavidin or by radioassay after secondarybinding of ¹²⁵I-streptavidin. Alternatively, GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B polypeptide is tagged with an antibody epitope at theN- or C-terminus as described supra with regard to polynucleotidesencoding polypeptides of the invention that are fused in frame to thecoding sequences for additional heterologous amino acid sequences.Binding of said epitope-tagged GMG-3, GMG-4, Cluster 1, GMG-6A, orGMG-6B polypeptide is detected with antibody specific for the epitope.

XIII. Cloning cDNA Encoding Cell Surface Receptor for GMG-3. GMG-4,Cluster 1, GMG-6A, or GMG-6B GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6BPolypeptide

The invention features methods of using GMG-3, GMG-4, Cluster 1, GMG-6A,or GMG-6B polypeptide to clone cDNA encoding a cell surface receptor forsaid GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide. Preferablysaid GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide comprisesall or part of the C-terminal globular C1q homology domain and has lipidpartitioning, lipid metabolism, or insulin-like activities.

In a preferred embodiment, said method of cloning a cell surfacereceptor for GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptidecomprises: isolating mRNA from a cell type expressing said cell surfacereceptor for GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide;converting said mRNA to cDNA; ligating said cDNA into a eukaryoticexpression vector containing the origin for SV40 replication;transiently transfecting pools of said ligated cDNA into COS cells usingdextran sulfate; culturing the transfected COS cells for about 48 h;detecting cell surface expression of said receptor for GMG-3, GMG-4,Cluster 1, GMG-6A, or GMG-6B polypeptide by contacting said transfectedCOS cells with biotinylated or epitope-tagged GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-4B polypeptide; contacting said biotinylated oreptiope-tagged GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptidebound to said transfected COS cells directly (biotinylated saidpolypeptide) or indirectly (epitope-tagged said polypeptide) with¹²⁵I-streptavidin; identifying said transfected COS cells labelled with¹²⁵I-streptavidin; recovering cDNA from said labelled COS cells; andrepeating said transient transfection with smaller pools of saidrecovered cDNA until transfection with a single clone of cDNA leads tocell surface expression of said receptor for GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B polypeptide. Said method of cloning cDNA encoding acell surface receptor for GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bpolypeptide by transient transfection of COS cells is well known tothose skilled in the art.

Other characteristics and advantages of the invention are described inthe Examples. These are meant to be exemplary only, and not to limit theinvention in any way. Throughout this application, various publications,patents and published patent applications are cited. The disclosures ofthese publications, patents and published patent specificationsreferenced in this application are hereby incorporated by reference intothe present disclosure.

EXAMPLES

The following Examples are provided for illustrative purposes and not asa means of limitation. One of ordinary skill in the art would be able todesign equivalent assays and methods based on the disclosure herein allof which form part of the instant invention.

Example 1 Northern Analysis of GMG-3, GMG-4 Cluster 1, GMG-6A, or GMG-6BDNA

Analysis of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B expression indifferent human tissues (adult and fetal) and cell lines, as well asmouse embryos in different stages of development, is accomplished byusing poly A⁺ RNA blots purchased from Clontech (e.g. #7780-1, 7757-1,7756-1, 7768-1 and 7763-1). Labeling of RNA probes is performed usingthe RNA Strip-EZ kit from Ambion as per manufacture's instructions.Hybridization of RNA probes to RNA blots is performed Ultrahybhybridization solution (Ambion). Briefly, blots are prehybridized for 30min at 58° C. (low-strigency) or 65° C. (high stringency). After addingthe labelled probe (2×10⁶ cpm/ml), blots are hybridized overnight (14-24hrs), and washed 2×20 min at 50° C. with 2×SSC/0.1% SDS (lowstringency), 2×20 min at 58° C. with 1×SSC/0.1% SDS (medium stringency)and 2×20 min at 65° C. with 1×SSC/0.1% SDS (high stringency). Afterwashings are completed blots are exposed on the phosphoimager (MolecularDynamics) for 1-3 days.

Example 2 In Vitro Tests of Metabolic-Related Activity

The activity of various preparations and various sequence variants ofGMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides are assessedusing various in vitro assays including those provided below. Theseassays are also exemplary of those that can be used to develop GMG-3,GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide antagonists andagonists. To do that, the effect of GMG-3, GMG-4, Cluster 1, GMG-6A, orGMG-6B polypeptides in the above assays, e.g. on leptin and/or LSRactivity, in the presence of the candidate molecules would be comparedwith the effect of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bpolypeptides in the assays in the absence of the candidate molecules.Since GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides reducebody weight in mice on a high-cafeteria diet (Example 5), these assaysalso serve to identify candidate treatments for reducing (or increasing)body weight.

Liver Cell Line:

Tests of efficacy of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bpolypeptides on LSR can be performed using liver cell lines, includingfor example, PLC, HepG2, Hep3B (human), Hepa 1-6, BPRCL (mouse), orMCA-RH777, MCA-RH8994 (rat).

BPRCL mouse liver cells (ATCC Repository) are plated at a density of300,000 cells/well in 6-well plates (day 0) in DMEM (high glucose)containing glutamine and penicillin-streptomycin (Bihain & Yen, 1992).Media is changed on day 2. On day 3, the confluent monolayers are washedonce with phosphate-buffered saline (PBS, pH 7.4) (2 mL/well). Cells areincubated at 37° C. for 30 min with increasing concentrations ofrecombinant GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide orGMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide fragmentcomprising the C-terminal globular C1q homology domain in DMEMcontaining 0.2% (w/v) BSA, 5 mM Hepes, 2 mM CaCl₂, 3.7 g/L sodiumbicarbonate, pH 7.5. Incubations are continued for 3 h at 37° C. afteraddition of 10 ng/mL ¹²⁵I-mouse leptin (specific activity, 22100cpm/ng). Monolayers are washed 2 times consecutively with PBS containing0.2% BSA, followed by 1 wash with PBS/BSA, and then 2 timesconsecutively with PBS. Cells are lysed with 0.1 N NaOH containing 0.24mM EDTA. Lysates are collected into tubes, and counted in agamma-counter.

Blood Brain Barrier Model:

The effect of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides onleptin transport in the brain can be determined using brain-derivedcells. One method that is envisioned is to use the blood/brain barriermodel described by Dehouck et al (J Neurochem 54:1798-801, 1990; herebyincorporated herein by reference in its entirety including any figures,tables, or drawings) that uses a co-culture of brain capillaryendothelial cells and astrocytes to test the effects of GMG-3, GMG-4,Cluster 1, GMG-6A, or GMG-6B polypeptides on leptin (or other molecules)transport via LSR or other receptors.

This assay would be an indicator of the potential effect of GMG-3,GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides on leptin transport tothe brain and could be used to screen GMG-3, GMG-4, Cluster 1, GMG-6A,or GMG-6B polypeptide variants for their ability to modulate leptintransport through LSR or other receptors in the brain. In addition,putative agonists and antagonists of the effect of GMG-3, GMG-4, Cluster1, GMG-6A, or GMG-6B polypeptides on leptin transport through LSR orother receptors could also be screened using this assay. Increasedtransport of leptin across the blood/brain barrier would presumablyincrease its action as a satiety factor.

FACS Analysis of LSR Expression

The effect of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides onLSR can also be determined by measuring the level of LSR expression atthe cell surface by flow surface cytometry, using anti-LSR antibodiesand fluorescent secondary antibodies. Flow cytometry is a laser-basedtechnology that is used to measure characteristics of biologicalparticles. The underlying principle of flow cytometry is that light isscattered and fluorescence is emitted as light from the excitationsource strikes the moving particles.

This is a high throughput assay that could be easily adapted to screenGMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides and variants aswell as putative agonists or antagonists of GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B polypeptides. Two assays are provided below. Theantibody, cell-line and GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bpolypeptide analogs would vary depending on the experiment, but a humancell-line, human anti-LSR antibody and GMG-3, GMG-4, Cluster 1, GMG-6A,or GMG-6B polypeptide fragment comprising the C-terminal globular C1qhomology domain could be used to screen for variants, agonists, andantagonists to be used to treat humans.

Assay 1:

Cells are pretreated with either intact GMG-3, GMG-4, Cluster 1, GMG-6A,or GMG-6B polypeptide or GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bpolypeptide fragment comprising the C-terminal globular C1q homologydomain (or untreated) before harvesting and analysis by FACS. Cells areharvested using non-enzymatic dissociation solution (Sigma), and thenare incubated for 1 h at 4° C. with a 1:200 dilution of anti-LSR 81B oran irrelevant anti-serum in PBS containing 1% (w/v) BSA. After washingtwice with the same buffer, goat anti-rabbit FITC-conjugated antibody(Rockland, Gilbertsville, Pa.) is added to the cells, followed by afurther incubation for 30 min at 4° C. After washing, the cells arefixed in 2% formalin. Flow cytometry analysis is done on a FACSCaliburcytometer (Becton-Dickinson, Franklin Lakes, N.J.).

Assay 2:

Cells are cultured in T175 flasks according to manufacturer'sinstructions for 48 hours prior to analysis.

Cells are washed once with FACS buffer (1×PBS/2% FBS, filtersterilized), and manually scraped from the flask in 10 mLs of FACSbuffer. The cell suspension is transferred to a 15 mL conical tube andcentrifuged at 1200 rpm, 4° C. for 5 minutes. Supernatant is discardedand cells are resuspended in 10 mL FACS buffer chilled to 4° C. A cellcount is performed and the cell density adjusted with FACS buffer to aconcentration of 1×10⁶ cells/mL. One milliliter of cell suspension wasadded to each well of a 48 well plate for analysis. Cells arecentrifuged at 1200 rpm for 5 minutes at 4° C. Plates are checked toensure that cells are pelleted, the supernatant is removed and cellsresuspended by running plate over a vortex mixer. One milliliter of FACSbuffer is added to each well, followed by centrifugation at 1200 rpm for5 minutes at 4° C. This described cell washing was performed a total of3 times.

Primary antibody, titered in screening experiments to determine properworking dilutions (for example 1:25, 1:50, 1:100, 1:200, 1:400, 1:500,1:800, 1:1000, 1:2000, 1:4000, 1:5000, or 1:10000), is added to cells ina total volume of 50 μL FACS buffer. Plates are incubated for 1 h at 4°C. protected from light. Following incubation, cells are washed 3 timesas directed above. Appropriate secondary antibody, titered in screeningexperiments to determine proper working dilutions (for example 1:25,1:50, 1:100, 1:200, 1:400, 1:500, 1:800, 1:1000, 1:2000, 1:4000, 1:5000,or 1:10000), is added to cells in a total volume of 50 μL FACS buffer.Plates are incubated for 1 h at 4° C. protected from light. Followingincubation, cells are washed 3 times as directed above. Upon final wash,cells are resuspended in 500 μL FACS buffer and transferred to a FACSacquisition tube. Samples are placed on ice protected from light andanalyzed within 1 hour.

Cellular Binding and Uptake of GMG-3, GMG-4, Cluster 1, GMG-6A, orGMG-6B Polypeptides as Detected by Fluorescence Microscopy

Fluorecein isothiocyanate (FITC) conjugation of GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B polypeptides: Purified GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B proteins at 1 mg/mL concentration are labelled withFITC using Sigma's FluoroTag FITC conjugation kit (Stock No. FITC-1).Protocol outlined in the Sigma Handbook for small-scale conjugation isfollowed for GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B proteinlabeling.

Cell Culture: C2C12 mouse skeletal muscle cells (ATCC, Manassas, Va.CRL-1772) and Hepa-1-6 mouse hepatocytes (ATCC, Manassas, Va. CRL-1830)are seeded into 6 well plates at a cell density of 2×10⁵ cells per well.C2C12 and Hepa-1-6 cells are cultured according to repository'sinstructions for 24-48 hours prior to analysis. Assay is performed whencells were 80% confluent.

FITC labelled GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B proteincellular binding and uptake using microscopy: C2C12 and Hepa 1-6 cellsare incubated in the presence/absence of antibody directed against humanLSR (81B: N-terminal sequence of human LSR; does not cross react withmouse LSR and 93A: c-terminal sequence, cross reacts with mouse LSR) oran antiserum directed against gC1qr (953) for 1 hour at 37° C., 5% CO2.LSR antibodies are added to the media at a concentration of 2 μg/mL. Theanti-gC1qr antiserum is added to the media at a volume of 2.5 μLundiluted serum (high concentration) or 1:100 dilution (lowconcentration). Following incubation with specified antibody,FITC-GMG-3, -GMG-4, -Cluster 1, -GMG-6A, or -GMG-6B polypeptide (50nM/mL) is added to each cell culture well. Cells are again incubated for1 hour at 37° C., 5% CO2. Cells are washed 2× with PBS, cells arescraped from well into 1 mL of PBS. Cell suspension is transferred to aneppendorf tube and centrifuged at 1000 rpm for 2 minutes. Supernatant isremoved and cells resuspended in 200 μL of PBS. Binding and uptake ofFITC-GMG-3, -GMG-4, -Cluster 1, -GMG-6A, or -GMG-6B polypeptide isanalyzed by fluorescence microscopy under 40× magnification.

This assay may be useful for identifying agents that facilitate orprevent the uptake and/or binding of GMG-3, GMG-4, Cluster 1, GMG-6A, orGMG-6B polypeptides to cells.

Effect on LSR as a Lipoprotein Receptor

The effect of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B protein on thelipoprotein binding, internalizing and degrading activity of LSR canalso be tested. Measurement of LSR as lipoprotein receptor is describedin Bihain & Yen, ((1992) Biochemistry 31:4628-36; hereby incorporatedherein in its entirety including any drawings, tables, or figures). Theeffect of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B protein on thelipoprotein binding, internalizing and degrading activity of LSR (orother receptors) can be compared with that of intact GMG-3, GMG-4,Cluster 1, GMG-6A, or GMG-6B protein, with untreated cells as anadditional control. This assay can also be used to screen for active andinhibitory variants of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bprotein, as well as agonists and antagonists of metabolic-relatedactivity.

Human liver PLC cells (ATCC Repository) are plated at a density of300,000 cells/well in 6-well plates (day 0) in DMEM (high glucose)containing glutamine and penicillin-streptomycin (Bihain & Yen, 1992).Media is changed on day 2. On day 3, the confluent monolayers are washedonce with phosphate-buffered saline (PBS, pH 7.4) (2 mL/well). Cells areincubated at 37° C. for 30 min with 10 ng/mL human recombinant leptin inDMEM containing 0.2% (w/v) BSA, 5 mM Hepes, 2 mM CaCl₂, 3.7 g/L sodiumbicarbonate, pH 7.5, followed by another 30 min incubation at 37° C.with increasing concentrations of GMG-3, GMG-4, Cluster 1, GMG-6A, orGMG-6B polypeptide. Incubations are continued for 2 h at 37° C. afteraddition of 0.8 mM oleate and 20 μg/mL ¹²⁵I-LDL. Monolayers are washed 2times consecutively with PBS containing 0.2% BSA, followed by 1 washwith PBS/BSA, and then 2 times consecutively with PBS. The amounts ofoleate-induced binding, uptake and degradation of ¹²⁵I-LDL are measuredas previously described (Bihain & Yen, 1992, supra). Results are shownas the mean of triplicate determinations.

GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B protein leads to an increasedactivity of LSR as a lipoprotein receptor. The oleate-induced bindingand uptake of LDL would be more affected by GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B protein as compared to the degradation. This increasedLSR activity would potentially result in an enhanced clearance oftriglyceride-rich lipoproteins during the postprandial state. Thus, moredietary fat would be removed through the liver, rather than beingdeposited in the adipose tissue.

This assay could be used to determine the efficiency of a compound (oragonists or antagonists) to increase or decrease LSR activity (orlipoprotein uptake, binding and degradation through other receptors),and thus affect the rate of clearance of triglyceride-rich lipoproteins.

Effect on Muscle Differentiation

C2C12 cells (murine skeletal muscle cell line; ATCC CRL 1772, Rockville,Md.) are seeded sparsely (about 15-20%) in complete DMEM (w/glutamine,pen/strep, etc)+10% FCS. Two days later they become 80-90% confluent. Atthis time, the media is changed to DMEM+2% horse serum to allowdifferentiation. The media is changed daily. Abundant myotube formationoccurs after 3-4 days of being in 2% horse serum, although the exacttime course of C2C12 differentiation depends on how long they have beenpassaged and how they have been maintained, among other things.

To test the effect of the presence of GMG-3, GMG-4, Cluster 1, GMG-6A,or GMG-6B protein on muscle differentiation, GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B polypeptide or polypeptide fragment comprising theC-terminal globular C1q homology domain (1 to 2.5 μg/mL) is added theday after seeding when the cells are still in DMEM w/ 10% FCS. Two daysafter plating the cells (one day after said GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B polypeptide or polypeptide fragment was first added),at about 80-90% confluency, the media is changed to DMEM+2% horse serumplus said GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide orpolypeptide fragment.

Effect on Muscle Cell Fatty Acid Oxidation

C2C12 cells are differentiated in the presence or absence of 2 μg/mLGMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B protein for 4 days. On day 4,oleate oxidation rates are determined by measuring conversion of1-¹⁴C-oleate (0.2 mM) to ¹⁴CO₂ for 90 min. This experiment can be usedto screen for active polypeptides and peptides as well as agonists andantagonists or activators and inhibitors of GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B polypeptides.

The effect of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide orpolypeptide fragment comprising the C-terminal globular C1q homologydomain on the rate of oleate oxidation can be compared in differentiatedC2C12 cells (murine skeletal muscle cells; ATCC, Manassas, Va. CRL-1772)and in a hepatocyte cell line (Hepa1-6; ATCC, Manassas, Va. CRL-1830).Cultured cells are maintained according to manufacturer's instructions.The oleate oxidation assay is performed as previously described (Muoioet al (1999) Biochem J 338;783-791). Briefly, nearly confluent myocytesare kept in low serum differentiation media (DMEM, 2.5% Horse serum) for4 days, at which time formation of myotubes became maximal. Hepatocytesare kept in the same DMEM medium supplemented with 10% FCS for 2 days.One hour prior to the experiment the media is removed and 1 mL ofpreincubation media (MEM, 2.5% Horse serum, 3 mM glucose, 4 mMGlutamine, 25 mM Hepes, 1% FFA free BSA, 0.25 mM Oleate, 5 μg/mLgentamycin) is added. At the start of the oxidation experiment ¹⁴C-Oleicacid (1 μCi/mL, American Radiolabelled Chemical Inc., St. Louis, Mo.) isadded and cells are incubated for 90 min at 37° C. in theabsence/presence of 2.5 μg/mL GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bpolypeptide or polypeptide fragment. After the incubation period 0.75 mLof the media is removed and assayed for ¹⁴C-oxidation products asdescribed below for the muscle FFA oxidation experiment.

Triglyceride and Protein Analysis Following Oleate Oxidation in CulturedCells

Following transfer of media for oleate oxidation assay, cells are placedon ice. To determine triglyceride and protein content, cells are washedwith 1 mL of 1×PBS to remove residual media. To each well 300 μL of celldissociation solution (Sigma) is added and incubated at 37° C. for 10min. Plates are tapped to loosen cells, and 0.5 mL of 1×PBS was added.The cell suspension is transferred to an eppendorf tube, each well isrinsed with an additional 0.5 mL of 1×PBS, and is transferred toappropriate eppendorf tube. Samples are centrifuged at 1000 rpm for 10minutes at room temperature. Supernatant is discarded and 750 μL of1×PBS/2% chaps is added to cell pellet. Cell suspension is vortexed andplaced on ice for 1 hour. Samples are then centrifuged at 13000 rpm for20 min at 4° C. Supernatants are transferred to new tube and frozen at−20° C. until analyzed. Quantitative measure of triglyceride level ineach sample is determined using Sigma Diagnostics GPO-TRINDER enzymatickit. The procedure outlined in the manual is adhered to, with thefollowing exceptions: assay is performed in 48 well plate, 350 μL ofsample volume was assayed, control blank consisted of 350 μL PBS/2%chaps, and standard contained 10 μL-standard provide in kit plus 690 μLPBS/2% chaps. Analysis of samples is carried out on a Packard SpectraCount at a wavelength of 550 mm Protein analysis is carried out on 25 μLof each supernatant sample using the BCA protein assay (Pierce)following manufacturer's instructions. Analysis of samples is carriedout on a Packard Spectra Count at a wavelength of 550 nm.

In Vitro Glucose Uptake by Muscle Cells

L6 Muscle cells are obtained from the European Culture Collection(Porton Down) and are used at passages 7-11. Cells are maintained instandard tissue culture medium DMEM, and glucose uptake is assessedusing [³H]-2-deoxyglucose (2DG) with or without GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B polypeptide fragment in the presence or absence ofinsulin (10⁻⁸ M) as has been previously described (Walker, P. S. et al.(1990) Glucose transport activity in L6 muscle cells is regulated by thecoordinate control of subcellular glucose transporter distribution,biosynthesis, and mRNA transcription. JBC 265(3):1516-1523; and Kilp, A.et al. (1992) Stimulation of hexose transport by metformin in L6 musclecells in culture. Endocrinology 130(5):2535-2544, which disclosures arehereby incorporated by reference in their entireties). Uptake of 2DG isexpressed as the percentage change compared with control (no addedinsulin or GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptidefragment). Values are presented as mean±SEM of sets of 4 wells perexperiment. Differences between sets of wells are evaluated by Student'st test, probability values p<0.05 are considered to be significant.

Example 3 Effect of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6BPolypeptides on Mice Fed a High-Fat Diet

Experiments are performed using approximately 6 week old C57B1/6 mice (8per group). All mice are housed individually. The mice are maintained ona high fat diet throughout each experiment. The high fat diet (cafeteriadiet; D12331 from Research Diets, Inc.) has the following composition:protein kcal % 16, sucrose kcal % 26, and fat kcal % 58. The fat isprimarily composed of coconut oil, hydrogenated.

After the mice are fed a high fat diet for 6 days, micro-osmotic pumpsare inserted using isoflurane anesthesia, and are used to providefull-length GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides,GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide fragments,saline, and an irrelevant peptide to the mice subcutaneously (s.c.) for18 days. GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides areprovided at doses of 100, 50, 25, and 2.5 μg/day and the irrelevantpeptide is provided at 10 μg/day. Body weight is measured on the first,third and fifth day of the high fat diet, and then daily after the startof treatment. Final blood samples are taken by cardiac puncture and areused to determine triglyceride (TG), total cholesterol (TC), glucose,leptin, and insulin levels. The amount of food consumed per day is alsodetermined for each group.

Example 4 Tests of Metabolic-Related Activity in Humans

Tests of the efficacy of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bpolypeptides in humans are performed in accordance with a physician'srecommendations and with established guidelines. The parameters testedin mice are also tested in humans (e.g. food intake, weight, TG, TC,glucose, insulin, leptin, FFA). It is expected that the physiologicalfactors would show changes over the short term. Changes in weight gainmight require a longer period of time. In addition, the diet would needto be carefully monitored. GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bpolypeptides, preferably GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bpolypeptides comprising the C-terminal globular C1q homology domain,would be given in daily doses of about 6 mg protein per 70 kg person orabout 10 mg per day. Other doses would also be tested, for instance 1 mgor 5 mg per day up to 20 mg, 50 mg, or 100 mg per day.

Example 5 Tests of Metabolic-Related Activity in a Murine LipoatrophicDiabetes Model

Previously, leptin was reported to reverse insulin resistance anddiabetes mellitus in mice with congenital lipodystrophy (Shimomura etal. Nature 401:73-76 (1999); hereby incorporated herein in its entiretyincluding any drawings, figures, or tables). Leptin was found to be lesseffective in a different lipodystrophic mouse model of lipoatrophicdiabetes (Gavrilova et al Nature 403: 850 (2000); hereby incorporatedherein in its entirety including any drawings, figures, or tables). Theinstant invention encompasses the use of GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B polypeptides for reducing the insulin resistance andhyperglycaemia in this model either alone or in combination with leptin,the leptin peptide (U.S. provisional application No. 60/155,506), orother compounds. Assays include that described previously in Gavrilovaet al. ((2000) Diabetes 49:1910-6; (2000) Nature 403:850) usingA-ZIP/F-1 mice, except that GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bpolypeptides would be administered using the methods previouslydescribed in Example 3 (or Examples 6-8). The glucose and insulin levelsof the mice would be tested, and the food intake and liver weightmonitored, as well as other factors, such as leptin, FFA, and TG levels,typically measured in our experiments (see Example 3, above, or Examples6-8).

Example 6 Effect of GMG-3, GMG-4Cluster 1, GMG-6A, or GMG-6BPolypeptides on Plasma Free Fatty Acid in C57 BL/6 Mice

The effect of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides onpostprandial lipemia (PPL) in normal C57BL6/J mice is tested.

The mice used in this experiment are fasted for 2 hours prior to theexperiment after which a baseline blood sample is taken. All bloodsamples are taken from the tail using EDTA coated capillary tubes (50 μLeach time point). At time 0 (8:30 AM), a standard high fat meal (6 gbutter, 6 g sunflower oil, 10 g nonfat dry milk, 10 g sucrose, 12 mLdistilled water prepared fresh following Nb#6, JF, pg. 1) is given bygavage (vol.=1% of body weight) to all animals.

Immediately following the high fat meal, 25 μg a GMG-3, GMG-4, Cluster1,GMG-6A, or GMG-6B polypeptide is injected i.p. in 100 μL saline. Thesame dose (25 μg/mL in 100 μL) is again injected at 45 min and at 1 hr45 min. Control animals are injected with saline (3×100 μL). Untreatedand treated animals are handled in an alternating mode.

Blood samples are taken in hourly intervals, and are immediately put onice; Plasma is prepared by centrifugation following each time point.Plasma is kept at −20° C. and free fatty acids (FFA), triglycerides (TG)and glucose are determined within 24 hours using standard test kits(Sigma and Wako). Due to the limited amount of plasma available, glucoseis determined in duplicate using pooled samples. For each time point,equal volumes of plasma from all 8 animals per treatment group arepooled.

Example 7 Effect of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6BPolypeptides on Plasma Leptin and Insulin in C57 BL/6 Mice

The effect of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides onplasma leptin and insulin levels during postprandial lipemia (PPL) innormal C57BL6/J mice is tested. The experimental procedure is the sameas that described in Example 6, except that blood was drawn only at 0, 2and 4 hours to allow for greater blood samples needed for thedetermination of leptin and insulin by RIA.

Briefly, 16 mice are fasted for 2 hours prior to the experiment afterwhich a baseline blood sample is taken. All blood samples are taken fromthe tail using EDTA coated capillary tubes (100 μL each time point). Attime 0 (9:00 AM), a standard high fat meal (see Example 6) is given bygavage (vol.=1% of body weight) to all animals. Immediately followingthe high fat meal, 25 μg of a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bpolypeptide is injected i.p. in 100 μL saline. The same dose (25 μg in100 μL) is again injected at 45 min and at 1 hr 45 min (treated group).Control animals are injected with saline (3×100 μL). Untreated andtreated animals are handled in an alternating mode.

Blood samples are immediately put on ice and plasma is prepared bycentrifugation following each time point. Plasma is kept at −20° C. andfree fatty acids (FFA) are determined within 24 hours using a standardtest kit (Wako). Leptin and Insulin are determined by RIA (ML-82K andSRI-13K, LINCO Research, Inc., St. Charles, Mo.) following themanufacturer's protocol; however; only 20 μL plasma is used. Eachdetermination is done in duplicate. Due to the limited amount of plasmaavailable, leptin and insulin are determined in 4 pools of 2 animalseach in both treatment groups.

Example 8 Effect of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6BPolypeptides on Plasma FFA, TG, and Glucose in C57 BL/6 Mice

The effect of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides onplasma FFA, TG, glucose, leptin and insulin levels during postprandiallipemia (PPL) in normal C57BL6/J mice has been described. Weight lossresulting from GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides(2.5 μg/day) given to normal C57BL6/J mice on a high fat diet has alsobeen shown (Example 3).

The experimental procedure is similar to that described in Example 6.Briefly, 14 mice re fasted for 2 hours prior to the experiment afterwhich a baseline blood sample is taken. All blood samples are taken fromthe tail using EDTA coated capillary tubes (50 μL each time point). Attime 0 (9:00 AM), a standard high fat meal (see Example 6) is given bygavage (vol.=1% of body weight) to all animals. Immediately followingthe high fat meal, 4 mice are injected 25 μg of a GMG-3, GMG-4, Cluster1, GMG-6A, or GMG-6B polypeptide i.p. in 100 μL saline. The same dose(25 μg in 100 μL) is again injected at 45 min and at 1 hr 45 min. Asecond treatment group receives 3 times 50 μg GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B polypeptide at the same intervals. Control animals areinjected with saline (3×100 μL). Untreated and treated animals arehandled in an alternating mode.

Blood samples are immediately put on ice. Plasma is prepared bycentrifugation following each time point. Plasma is kept at −20° C. andfree fatty acids (FFA), triglycerides (TG) and glucose are determinedwithin 24 hours using standard test kits (Sigma and Wako).

Example 9 Effect of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6BPolypeptides on FFA Following Epinephrine Injection

In mice, plasma free fatty acids increase after intragastricadministration of a high fat/sucrose test meal. These free fatty acidsare mostly produced by the activity of lipolytic enzymes i.e.lipoprotein lipase (LPL) and hepatic lipase (HL). In this species, theseenzymes are found in significant amounts both bound to endothelium andfreely circulating in plasma. Another source of plasma free fatty acidsis hormone sensitive lipase (HSL) that releases free fatty acids fromadipose tissue after β-adrenergic stimulation. To test whether GMG-3,GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides also regulate themetabolism of free fatty acid released by HSL, mice are injected withepinephrine.

Two groups of mice are given epinephrine (5 μg) by intraperitonealinjection. A treated group is injected with a GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B polypeptide (25 μg) one hour before and again togetherwith epinephrine, while control animals receive saline. Plasma isisolated and free fatty acids and glucose are measured as describedabove (Example 8).

Example 10 Effect of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6BPolypeptides on Muscle FFA Oxidation

To investigate the effect of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bpolypeptides on muscle free fatty acid oxidation, intact hind limbmuscles from C57BL/6J mice are isolated and FFA oxidation is measuredusing oleate as substrate (Clee, S. M. et al. Plasma and vessel walllipoprotein lipase have different roles in atherosclerosis. J Lipid Res41, 521-531 (2000); Muoio, D. M., Dohm, G. L., Tapscott, E. B. &Coleman, R. A. Leptin opposes insulin's effects on fatty acidpartitioning in muscles isolated from obese ob/ob mice. Am J Physiol276, E913-921 (1999)) Oleate oxidation in isolated muscle is measured aspreviously described (Cuendet et al (1976) J Clin Invest 58:1078-1088;Le Marchand-Brustel, Y., Jeanrenaud, B. & Freychet, P. Insulin bindingand effects in isolated soleus muscle of lean and obese mice. Am JPhysiol 234, E348-E358 (1978). Briefly, mice are sacrificed by cervicaldislocation and soleus and EDL muscles are rapidly isolated from thehind limbs. The distal tendon of each muscle is tied to a piece ofsuture to facilitate transfer among different media. All incubations arecarried out at 30° C. in 1.5 mL of Krebs-Henseleit bicarbonate buffer(118.6 mM NaCl, 4.76 mM KCl, 1.19 mM KH₂PO₄, 1.19 mM MgSO₄, 2.54 mMCaCl₂, 25 mM NaHCO₃, 10 mM Hepes, pH 7.4) supplemented with 4% FFA freebovine serum albumin (fraction V, RIA grade, Sigma) and 5 mM glucose(Sigma). The total concentration of oleate (Sigma) throughout theexperiment is 0.25 mM. All media are oxygenated (95% O₂; 5% CO₂) priorto incubation. The gas mixture is hydrated throughout the experiment bybubbling through a gas washer (Kontes Inc., Vineland, N.J.).

Muscles are rinsed for 30 min in incubation media with oxygenation. Themuscles are then transferred to fresh media (1.5 mL) and incubated at30° C. in the presence of 1 μCi/mL [1-¹⁴C] oleic acid (AmericanRadiolabelled Chemicals). The incubation vials containing this media aresealed with a rubber septum from which a center well carrying a piece ofWhatman paper (1.5 cm×11.5 cm) is suspended.

After an initial incubation period of 10 min with constant oxygenation,gas circulation is removed to close the system to the outsideenvironment and the muscles are incubated for 90 min at 30° C. At theend of this period, 0.45 mL of Solvable (Packard Instruments, Meriden,Conn.) is injected onto the Whatman paper in the center well and oleateoxidation by the muscle is stopped by transferring the vial onto ice.

After 5 min, the muscle is removed from the medium, and an aliquot of0.5 mL medium is also removed. The vials are closed again and 1 mL of35% perchloric acid is injected with a syringe into the media bypiercing through the rubber septum. The CO₂ released from the acidifiedmedia is collected by the Solvable in the center well. After a 90 mincollection period at 30° C., the Whatman paper is removed from thecenter well and placed in scintillation vials containing 15 mL ofscintillation fluid (HionicFlour, Packard Instruments, Meriden, Conn.).The amount of ¹⁴C radioactivity is quantitated by liquid scintillationcounting. The rate of oleate oxidation is expressed as nmol oleateproduced in 90 min/g muscle.

To test the effect of full-length GMG-3, GMG-4, Cluster 1, GMG-6A, orGMG-6B polypeptide or GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6Bpolypeptide fragment comprising the C-terminal globular C1q homologydomain on oleate oxidation, these proteins are added to the media at afinal concentration of 2.5 μg/mL and maintained in the media throughoutthe procedure.

Example 11 Effect of GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6BPolypeptides on Triglyceride in Muscle & Liver Isolated from Mice

To determine whether the increased FFA oxidation induced by GMG-3,GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides is also accompanied byincreased FFA delivery into muscle or liver, the hindlimb muscle andliver triglyceride content is measured after the GMG-3, GMG-4, Cluster1, GMG-6A, or GMG-6B polypeptide treatment of mice. Hind limb muscles aswell as liver samples are removed from treated and untreated animals andthe triglyceride and free fatty acid concentration is determinedfollowing a standard lipid extraction method (Shimabukuro, M. et al.Direct antidiabetic effect of leptin through triglyceride depletion oftissues. Proc Natl Acad Sci USA 94:4637-4641 (1997)) followed by TG andFFA analysis using standard test kits.

Example 12 Effect of GMG-3, GMG-4, Cluster 1, GMG-6A or GMG-6BPolypeptides on FFA Following Intralipid Injection

Two groups of mice are intravenously (tail vein) injected with 30 μLbolus of Intralipid-20% (Clintec) to generate a sudden rise in plasmaFFAs, thus by-passing intestinal absorption. (Intralipid is anintravenous fat emulsion used in nutritional therapy). A treated group(GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide-treated) isinjected with a GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptide(25 μg) at 30 and 60 minutes before Intralipid is given, while controlanimals (□ control) received saline. Plasma is isolated and FFAs aremeasured as described previously. The effect of GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B polypeptides on the decay in plasma FFAs following thepeak induced by Intralipid injection is then monitored.

Example 13 In Vitro Glucose Uptake by Muscle Cells

L6 Muscle cells are obtained from the European Culture Collection(Porton Down) and are used at passages 7-11. Cells are maintained instandard tissue culture medium DMEM, and glucose uptake is assessedusing [³H]-2-deoxyglucose (2DG) with or without GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B polypeptides in the presence or absence of insulin(10⁻⁸ M) as has been previously described (Walker, P. S. et al. (1990)Glucose transport activity in L6 muscle cells is regulated by thecoordinate control of subcellular glucose transporter distribution,biosynthesis, and mRNA transcription. JBC 265:1516-1523; and Kilp, A. etal. (1992) Stimulation of hexose transport by metformin in L6 musclecells in culture, Endocrinology 130:2535-2544, which disclosures arehereby incorporated by reference in their entireties). Uptake of 2DG isexpressed as the percentage change compared with control (no addedinsulin or GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B). Values arepresented as mean±SEM of sets of 4 wells per experiment. Differencesbetween sets of wells are evaluated by Student's t test, probabilityvalues p<0.05 are considered to be significant.

Example 14 In Vivo Tests for Metabolic-Related Activity in RodentDiabetes Models

As metabolic profiles differ among various animal models of obesity anddiabetes, analysis of multiple models is undertaken to separate theeffects GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides onhyperglycemia, hyperinsulinemia, hyperlipidemia and obesity. Mutationswithin colonies of laboratory animals and different sensitivities todietary regimens have made the development of animal models withnon-insulin dependent diabetes associated with obesity and insulinresistance possible. Genetic models such as db/db and ob/ob (SeeDiabetes, (1982) 31(1): 1-6) in mice and fa/fa in zucker rats have beendeveloped by the various laboratories for understanding thepathophysiology of disease and testing the efficacy of new antidiabeticcompounds (Diabetes, (1983) 32: 830-838; Annu Rep Sankyo Res Lab (1994)46: 1-57). The homozygous animals, C57 BL/KsJ-db/db mice developed byJackson Laboratory, US, are obese, hyperglycemic, hyperinsulinemic andinsulin resistant (J Clin Invest, (1990) 85: 962-967), whereasheterozygous are lean and normoglycemic. In db/db model, mouseprogressively develops insulinopenia with age, a feature commonlyobserved in late stages of human type II diabetes when blood sugarlevels are insufficiently controlled. The state of pancreas and itscourse vary according to the models. Since this model resembles that oftype II diabetes mellitus, the compounds of the present invention aretested for blood sugar and triglycerides lowering activities. Zucker(fa/fa) rats are severely obese, hyperinsulinemic, and insulin resistant(Coleman, Diabetes 31:1, 1982; E. Shafrir, in Diabetes Mellitus; H.Rifkin and D. Porte, Jr. Eds. (Elsevier Science Publishing Co., Inc.,New York, ed. 4, 1990), pp. 299-340), and the fa/fa mutation may be therat equivalent of the murine db mutation (Friedman et al., Cell69:217-220, 1992; Truett et al., Proc. Natl. Acad. Sci. USA 88:7806,1991). Tubby (tub/tub) mice are characterized by obesity, moderateinsulin resistance and hyperinsulinemia without significanthyperglycemia (Coleman et al., J. Heredity 81:424, 1990).

Previously, leptin was reported to reverse insulin resistance anddiabetes mellitus in mice with congenital lipodystrophy (Shimomura etal. Nature 401: 73-76 (1999). Leptin is found to be less effective in adifferent lipodystrophic mouse model of lipoatrophic diabetes (Gavrilovaet al Nature 403: 850 (2000); hereby incorporated herein in its entiretyincluding any drawings, figures, or tables).

The streptozotocin (STZ) model for chemically-induced diabetes is testedto examine the effects of hyperglycemia in the absence of obesity.STZ-treated animals are deficient in insulin and severely hyperglycemic(Coleman, Diabetes 31:1, 1982; E. Shafrir, in Diabetes Mellitus; H.Rifkin and D. Porte, Jr. Eds. (Elsevier Science Publishing Co., Inc.,New York, ed. 4, 1990), pp. 299-340). The monosodium glutamate (MSG)model for chemically-induced obesity (Olney, Science 164:719, 1969;Cameron et al., Clin Exp Pharmacol Physiol 5:41, 1978), in which obesityis less severe than in the genetic models and develops withouthyperphagia, hyperinsulinemia and insulin resistance, is also examined.Finally, a non-chemical, non-genetic model for induction of obesityincludes feeding rodents a high fat/high carbohydrate (cafeteria diet)diet ad libitum.

The instant invention encompasses the use of GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B polypeptides for reducing the insulin resistance andhyperglycemia in any or all of the above rodent diabetes models or inhumans with Type I or Type II diabetes or other prefered metabolicdiseases described previously or models based on other mammals. In thecompositions of the present invention the GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B polypeptides may, if desired, be associated with othercompatible pharmacologically active antidiabetic agents such as insulin,leptin (U.S. provisional application No. 60/155,506), or troglitazone,either alone or in combination. Assays include that described previouslyin Gavrilova et al. ((2000) Diabetes 49:1910-6; (2000) Nature 403:850)using A-ZIP/F-1 mice, except that GMG-3, GMG-4, Cluster 1, GMG-6A, orGMG-6B polypeptides are administered intraperitoneally, subcutaneously,intramuscularly or intravenously. The glucose and insulin levels of themice would be tested, and the food intake and liver weight monitored, aswell as other factors, such as leptin, FFA, and TG levels, typicallymeasured in our experiments.

In Vivo Assay for Anti-Hyperglycemic Activity of GMG-3, GMG-4, Cluster1, GMG-6A, or GMG-6B Polypeptides

Genetically altered obese diabetic mice (db/db) (male, 7-9 weeks old)are housed (7-9 mice/cage) under standard laboratory conditions at 22°C. and 50% relative humidity, and maintained on a diet of Purina rodentchow and water ad libitum. Prior to treatment, blood is collected fromthe tail vein of each animal and blood glucose concentrations aredetermined using One Touch Basic Glucose Monitor System (Lifescan). Micethat have plasma glucose levels between 250 to 500 mg/dl are used. Eachtreatment group consists of seven mice that are distributed so that themean glucose levels are equivalent in each group at the start of thestudy. db/db mice are dosed by micro-osmotic pumps, inserted usingisoflurane anesthesia, to provide GMG-3, GMG-4, Cluster 1, GMG-6A, orGMG-6B polypeptides, saline, and an irrelevant peptide to the micesubcutaneously (s.c.). Blood is sampled from the tail vein hourly for 4hours and at 24, 30 h post-dosing and analyzed for blood glucoseconcentrations. Food is withdrawn from 0-4 h post dosing andreintroduced thereafter. Individual body weights and mean foodconsumption (each cage) are also measured after 24 h. Significantdifferences between groups (comparing GMG-3, GMG-4, Cluster 1, GMG-6A,or GMG-6B treated to saline-treated) are evaluated using Student t-test.

In Vivo Insulin Sensitivity Assay

In vivo insulin sensitivity is examined by utilizing two-stephyperinsulinemic-euglycemic clamps according to the following protocol.Rodents from any or all of the various models described in Example 2 arehoused for at least a week prior to experimental procedures. Surgeriesfor the placement of jugular vein and carotid artery catheters areperformed under sterile conditions using ketamine and xylazine (i.m.)anesthesia. After surgery, all rodents are allowed to regainconsciousness and placed in individual cages. GMG-3, GMG-4, Cluster 1,GMG-6A, or GMG-6B polypeptides or vehicle is administered through thejugular vein after complete recovery and for the following two days.Sixteen hours after the last treatment, hyperinsulinemic-euglycemicclamps are performed. Rodents are placed in restrainers and a bolus of 4μCi [3-³H] glucose (NEN) is administered, followed by a continuousinfusion of the tracer at a dose of 0.2 μCi/min (20 μl/min). Two hoursafter the start of the tracer infusion, 3 blood samples (0.3 ml each)are collected at 10 minute intervals (−20-0 min) for basal measurements.An insulin infusion is then started (5 mU/kg/min), and 100 μl bloodsamples are taken every 10 min. to monitor plasma glucose. A 30% glucosesolution is infused using a second pump based on the plasma glucoselevels in order to reach and maintain euglycemia. Once a steady state isestablished at 5 mU/kg/min insulin (stable glucose infusion rate andplasma glucose), 3 additional blood samples (0.3 ml each) are obtainedfor measurements of glucose, [3-³H] glucose and insulin (100-120 min.).A higher dose of insulin (25 mU/kg/min.) is then administered andglucose infusion rates are adjusted for the second euglycemic clamp andblood samples are taken at min. 220-240. Glucose specific activity isdetermined in deproteinized plasma and the calculations of Rd andhepatic glucose output (HGO) are made, as described (Lang et al.,Endocrinology 130:43, 1992). Plasma insulin levels at basal period andafter 5 and 25 mU/kg/min, infusions are then determined and comparedbetween GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B treated and vehicletreated rodents.

Insulin regulation of glucose homeostasis has two major components;stimulation of peripheral glucose uptake and suppression of hepaticglucose output. Using tracer studies in the glucose clamps, it ispossible to determine which portion of the insulin response is affectedby the GMG-3, GMG-4, Cluster 1, GMG-6A, or GMG-6B polypeptides.

1. A method of treating an obesity-related disease or disordercomprising administering to an individual a composition comprising apolypeptide or biologically active fragment thereof, wherein saidpolypeptide or biologically active fragment thereof comprises all orpart of a C-terminal globular C1q homology domain and wherein saidpolypeptide comprises SEQ ID NO: 2 or comprises a biologically activefragment of SEQ ID NO:
 2. 2. The method of claim 1, wherein saidobesity-related disease or disorder is selected from the groupconsisting of: (a) obesity; (b) impaired glucose tolerance; (c) insulinresistance; (d) Syndrome X; and (e) Type II diabetes.
 3. The method ofclaim 2, wherein said obesity-related disease or disorder is obesity. 4.The method of claim 2, wherein said obesity-related disease is impairedglucose tolerance.
 5. The method of claim 2, wherein saidobesity-related disease is insulin resistance.
 6. The method of claim 2,wherein said obesity-related disease is Syndrome X.
 7. The method ofclaim 2, wherein said obesity-related disease is Type II diabetes. 8.The method of claim 1, wherein said biologically active polypeptidefragment is selected from the group consisting of: (a) amino acids20-333 of SEQ ID NO: 2; (b) amino acids 188-333 of SEQ ID NO: 2; (c)amino acids 191-333 of SEQ ID NO: 2; (d) amino acids 193-333 of SEQ IDNO: 2; and (e) amino acids 252-317 of SEQ ID NO:
 2. 9. The method ofclaim 8, wherein said biologically active polypeptide fragment comprisesamino acids 20-333 of SEQ ID NO:
 2. 10. The method of claim 8, whereinsaid biologically active polypeptide fragment comprises amino acids188-333 of SEQ ID NO:
 2. 11. The method of claim 8, wherein saidbiologically active polypeptide fragment comprises amino acids 191-333of SEQ ID NO:
 2. 12. The method of claim 8, wherein said biologicallyactive polypeptide fragment comprises amino acids 193-333 of SEQ ID NO:2.
 13. The method of claim 8, wherein said biologically activepolypeptide fragment comprises amino acids 252-317 of SEQ ID NO:
 2. 14.The method of claim 1, wherein said polypeptide or biologically activefragment thereof is administered prior to the onset of clinical symptomsassociated with said obesity-related disease or disorder.